Bacterial host strain

ABSTRACT

A recombinant gram-negative bacterial cell comprising one or more of the following mutated protease genes: a) a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene; b) a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene; and c) a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; wherein the cell is isogenic to a wild-type bacterial cell except for the mutated Tsp gene and/or mutated ptr gene and/or mutated DegP gene and optionally a polynucleotide sequence encoding a protein of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/827,408, filedAug. 17, 2015, now U.S. Pat. No. 9,315,370, which is a continuation ofU.S. Ser. No. 13/497,987, filed Jun. 29, 2012, now U.S. Pat. No.9,109,216, which is the U.S. national stage application of InternationalPatent Application No. PCT/GB2010/001790, filed Sep. 23, 2010, thedisclosures of which are hereby incorporated by reference in theirentirety, including all figures, tables and amino acid or nucleic acidsequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Feb. 20, 2015 and is 49 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

The invention relates to a recombinant bacterial host strain,particularly E. coli. The invention also relates to a method forproducing a protein of interest in such a cell.

BACKGROUND OF THE INVENTION

Bacterial cells, such as E. coli, are commonly used for producingrecombinant proteins. There are many advantages to using bacterialcells, such as E. coli, for producing recombinant proteins particularlydue to the versatile nature of bacterial cells as host cells allowingthe gene insertion via plasmids. E. coli have been used to produce manyrecombinant proteins including human insulin.

Despite the many advantages to using bacterial cells to producerecombinant proteins, there are still significant limitations includingthe difficulty of producing protease sensitive proteins. Proteases playan important role in turning over old and miss-folded proteins in the E.coli periplasm and cytoplasm. Bacterial proteases act to degrade therecombinant protein of interest, thereby often significantly reducingthe yield of active protein.

A number of bacterial proteases have been identified. In E. coliproteases including Protease III (ptr), DegP, OmpT, Tsp, prlC, ptrA,ptrB, pepA-T, tsh, espc, eatA, clpP and lon have been identified.

The Protease III (ptr) protein is a 110 kDa periplasmic protease whichdegrades high molecular weight proteins.

Tsp (also known as Prc) is a 60 kDa periplasmic protease. The firstknown substrate of Tsp was Penicillin-binding protein-3 (PBP3)(Determination of the cleavage site involved in C-terminal processing ofpenicillin-binding protein 3 of Escherichia coli; Nagasawa H, SakagamiY, Suzuki A, Suzuki H, Hara H, Hirota Y. J Bacteriol. 1989 November;171(11):5890-3 and Cloning, mapping and characterization of theEscherichia coli Tsp gene which is involved in C-terminal processing ofpenicillin-binding protein 3; Hara H, Yamamoto Y, Higashitani A, SuzukiH, Nishimura Y. J Bacteriol. 1991 August; 173 (15):4799-813) but it waslater discovered that the Tsp was also able to cleave phage tailproteins and, therefore, it was renamed as Tail Specific Protease (Tsp)(Silber et al., Proc. Natl. Acad. Sci. USA, 89: 295-299 (1992)). Silberet al. (Deletion of the prc(tsp) gene provides evidence for additionaltail-specific proteolytic activity in Escherichia coli K-12; Silber, K.R., Sauer, R. T.; Mol Gen Genet 1994 242:237-240) describes a prcdeletion strain (KS1000) wherein the mutation was created by replacing asegment of the prc gene with a fragment comprising a Kan^(r) marker.

DegP (also known as HtrA) is a 46 kDa protein having dual function as achaperone and a protease (Families of serine peptidases; Rawlings N D,Barrett A J. Methods Enzymol. 1994; 244:19-61).

It is known to knockout bacterial proteases in order to affect the yieldof recombinant protein.

Georgiou et al. (Construction and characterization of Escherichia colistrains deficient in multiple secreted proteases: protease III degradeshigh-molecular-weight substrates in vivo. Baneyx F, Georgiou G. JBacteriol. 1991 April; 173(8):2696-703) studied the effects on growthproperties and protein stability of E. coli strains deficient inprotease III constructed by insertional inactivation of the ptr gene andobserved an increase in the expression of a protease-sensitive secretedpolypeptide. A strain comprising the ptr mutation and also deficient inthe secreted protease DegP was also produced and found to have adecreased growth rate and an increase in protein expression. In Georgiouet al., the E. coli strains deficient in protease III and/or DegP wereconstructed from the KS272 parental strain which already comprises anumber of genomic mutations.

U.S. Pat. No. 5,264,365 (Georgiou et al.) discloses the construction ofprotease-deficient Escherichia coli hosts which when combined with anexpression system are useful for the production of proteolyticallysensitive polypeptides.

Meerman et al. (Construction and characterization of Escherichia colistrains deficient in All Known Loci Affecting the Proteolytic Stabilityof Secreted Recombinant Proteins. Meerman H. J., Georgeou G., NatureBiotechnology, 1994 November; 12; 1107-1110) disclose E. coli strainscomprising mutations in the rpoH, the RNA polymerase sigma factorresponsible for heat shock protein synthesis, and different combinationsof mutations in protease genes including DegP, Protease III, Tsp(Prc)and OmpT, where null mutations of the protease genes were caused byinsertional mutations. In Meerman et al., the E. coli strains deficientin one or more of Tsp, protease III and DegP were constructed from theKS272 parental strain which already comprises a number of genomicmutations.

U.S. Pat. No. 5,508,192 (Georgiou et al.) discloses a method ofproducing recombinant polypeptides in protease-deficient bacterial hostsand constructs of single, double, triple and quadruple proteasedeficient bacteria which also carry a mutation in the rpoH gene.

Chen et al describes the construction of E. coli strains carryingdifferent combinations of mutations in prc (Tsp) and DegP created byamplifying the upstream and downstream regions of the gene and ligatingthese together on a vector comprising selection markers and a sprW148Rmutation (High-level accumulation of a recombinant antibody fragment inthe periplasm of Escherichia coli requires a triple-mutant (ΔDegP Δprcspr W148R) host strain. Chen C, Snedecor B, Nishihara J C, Joly J C,McFarland N, Andersen D C, Battersby J E, Champion K M. BiotechnolBioeng. 2004 Mar. 5; 85(5):463-74). The combination of the ΔDegP, Δprcand W148Rspr mutations were found to provide the highest levels ofantibody light chain, antibody heavy chain and F(ab′)2-LZ. EP1341899discloses an E. coli strain that is deficient in chromosomal DegP andprc encoding proteases DegP and Prc, respectively, and harbors a mutantspr gene that encodes a protein that suppresses growth phenotypesexhibited by strains harboring prc mutants.

Kandilogiannaki et al (Expression of a recombinant human anti-MUC1 scFvfragment in protease-deficient Escherichia coli mutants. KandilogiannakiM, Koutsoudakis G, Zafiropoulos A, Krambovitis E. Int J Mol Med. 2001June; 7(6):659-64) describes the utilization of a protease deficientstrain for the expression of a scFv protein.

The protease deficient bacterial strains used previously to expressrecombinant proteins comprise further mutations of genes involved incell metabolism and DNA replication such as, for example phoA, fhuA,lac, rec, gal, ara, arg, thi and pro in E. coli strains. These mutationsmay have many deleterious effects on the host cell including effects oncell growth, stability, recombinant protein expression yield andtoxicity. Strains having one or more of these genomic mutations,particularly strains having a high number of these mutations, mayexhibit a loss of fitness which reduces bacterial growth rate to a levelwhich is not suitable for industrial protein production. Further, any ofthe above genomic mutations may affect other genes in cis and/or intrans in unpredictable harmful ways thereby altering the strain'sphenotype, fitness and protein profile. Further, the use of heavilymutated cells is not generally suitable for producing recombinantproteins for commercial use, particularly therapeutics, because suchstrains generally have defective metabolic pathways and hence may growpoorly or not at all in minimal or chemically defined media.

Protease deficient bacterial strains also typically comprise knockoutmutations to one or more protease encoding genes which have been createdby insertion of a DNA sequence into the gene coding sequence. Theinserted DNA sequence typically codes for a selection marker such as anantibiotic resistance gene. Whilst this mutation method may be effectiveat knocking out the target protease, there are many disadvantagesassociated with this method. One disadvantage is the insertion of theforeign DNA, such as an antibiotic resistance gene, causes disruption inthe host's genome which may result in any number of unwanted effectsincluding the over-expression of detrimental proteins and/ordown-regulation or knockout of other essential proteins. This effect isparticularly evident for those genes positioned immediately upstream ordownstream of the target protease gene. A further disadvantage to theinsertion of foreign DNA, particularly antibiotic resistance genes, isthe unknown phenotypic modifications to the host cell which may affectexpression of the target protein and/or growth of the host cell and mayalso make the host cell unsuitable for production of proteins intendedfor use as therapeutics. Antibiotic resistance proteins are particularlydisadvantageous for biosafety requirements large scale manufacturingparticularly for the production of therapeutics for humanadministration. A further disadvantage to the insertion of antibioticresistance markers is the metabolic burden on the cell created by theexpression of the protein encoded by the antibiotic resistance gene. Theuse of antibiotic resistance markers for use as markers for geneticmanipulations such as knockout mutations, are also limited by the numberof different antibiotic resistance markers available.

Accordingly, there is still a need to provide new bacterial strainswhich provide advantageous means for producing recombinant proteins.

SUMMARY OF THE INVENTION

It is an aim of the present invention to solve one or more of theproblems described above.

In a first aspect the present invention provides a recombinantgram-negative bacterial cell comprising one or more of the followingmutated protease genes:

-   -   a. a mutated Tsp gene, wherein the mutated Tsp gene encodes a        Tsp protein having reduced protease activity or is a knockout        mutated Tsp gene;    -   b. a mutated ptr gene, wherein the mutated ptr gene encodes a        Protease III protein having reduced protease activity or is a        knockout mutated ptr gene; and    -   c. a mutated DegP gene encoding a DegP protein having chaperone        activity and reduced protease activity;

wherein the cell is isogenic to a wild-type bacterial cell except forthe mutated Tsp gene and/or mutated ptr gene and/or mutated DegP geneand optionally a polynucleotide sequence encoding a protein of interest.

In one embodiment the present invention provides a cell comprising amutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp proteinhaving reduced protease activity or is a knockout mutated Tsp gene andno further mutated protease genes. Accordingly, the present inventionprovides a cell which is isogenic to a wild-type bacterial cell exceptfor the mutated Tsp gene and optionally a polynucleotide sequenceencoding a protein of interest.

In one embodiment the present invention provides a cell comprising amutated ptr gene, wherein the mutated ptr gene encodes a Protease IIIprotein having reduced protease activity or is a knockout mutated ptrgene and no further mutated protease genes. Accordingly, the presentinvention provides a cell which is isogenic to a wild-type bacterialcell except for the mutated ptr gene and optionally a polynucleotidesequence encoding a protein of interest.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity and no further mutated protease genes.Accordingly, the present invention provides a cell which is isogenic toa wild-type bacterial cell except for the mutated DegP gene andoptionally a polynucleotide sequence encoding a protein of interest.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, a mutated Tsp gene, wherein the mutated Tspgene encodes a Tsp protein having reduced protease activity or is aknockout mutated Tsp gene and no further mutated protease genes.Accordingly, the present invention provides a cell which is isogenic toa wild-type bacterial cell except for the mutated DegP gene and themutated Tsp gene and optionally a polynucleotide sequence encoding aprotein of interest.

In one embodiment the present invention provides a cell comprising amutated ptr gene, wherein the mutated ptr gene encodes a Protease IIIprotein having reduced protease activity or is a knockout mutated ptrgene, a mutated Tsp gene wherein the mutated Tsp gene encodes a Tspprotein having reduced protease activity or is a knockout mutated Tspgene and no further mutated protease genes. Accordingly, the presentinvention provides a cell which is isogenic to a wild-type bacterialcell except for the mutated ptr gene and the mutated Tsp gene andoptionally a polynucleotide sequence encoding a protein of interest.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, a mutated ptr gene, wherein the mutated ptrgene encodes a Protease III protein having reduced protease activity oris a knockout mutated ptr gene and no further mutated protease genes.Accordingly, the present invention provides a cell which is isogenic toa wild-type bacterial cell except for the mutated DegP gene and mutatedptr gene and optionally a polynucleotide sequence encoding a protein ofinterest.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, a mutated ptr gene, wherein the mutated ptrgene encodes a Protease III protein having reduced protease activity oris a knockout mutated ptr gene, a mutated Tsp gene, wherein the mutatedTsp gene encodes a Tsp protein having reduced protease activity or is aknockout mutated Tsp gene and no further mutated protease genes.Accordingly, the present invention provides a cell which is isogenic toa wild-type bacterial cell except for the mutated DegP gene, the mutatedptr gene and the mutated Tsp gene and optionally a polynucleotidesequence encoding a protein of interest.

In a preferred embodiment the mutated ptr gene and/or the mutated Tspgene referred to above are knockout mutations.

The present inventors have found that a bacterial host strain isogenicto a wild-type bacterial cell except for one or more of the abovemutated protease provides an advantageous host for producing arecombinant protein of interest. The cells provided by the presentinvention have reduced protease activity compared to a non-mutated cell,which may reduce proteolysis of a recombinant protein of interest,particularly proteins of interest which are proteolytically sensitive.In addition, the cell according to the present invention carries onlyminimal mutations to the genomic sequence in order to introduce one ormore of the above protease mutations and does not carry any othermutations which may have deleterious effects on the cell's growth and/orability to express a protein of interest.

One or more of the gram-negative cells provided by the present inventionmay provide a high yield of the recombinant protein of interest. One ormore of the gram-negative cells provided by the present invention mayprovide a fast rate of production of a protein of interest. One or moreof the cells may provide fast initial yield of the recombinant proteinof interest. Further, one or more of the cells may show good growthcharacteristics.

In a second aspect, the present invention provides a recombinantgram-negative bacterial cell comprising:

-   -   a. a knockout mutated Tsp gene comprising a mutation to the gene        start codon and/or one or more stop codons positioned downstream        of the gene start codon and upstream of the gene stop codon;        and/or    -   b. a knockout mutated ptr gene comprising a mutation to the gene        start codon and/or one or more stop codons positioned downstream        of the gene start codon and upstream of the gene stop codon; and    -   c. optionally a mutated DegP gene encoding a DegP protein having        chaperone activity and reduced protease activity.

In one embodiment the present invention provides a cell comprising aknockout mutated Tsp gene comprising a mutation to the gene start codonand/or one or more stop codons positioned downstream of the gene startcodon and upstream of the gene stop codon.

In one embodiment the present invention provides a cell comprising aknockout mutated ptr gene comprising a mutation to the gene start codonand/or one or more stop codons positioned downstream of the gene startcodon and upstream of the gene stop codon.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity and a knockout mutated Tsp gene comprising amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon.

In one embodiment the present invention provides a cell comprising aknockout mutated ptr gene comprising a mutation to the gene start codonand/or one or more stop codons positioned downstream of the gene startcodon and upstream of the gene stop codon and a knockout mutated Tspgene comprising a mutation to the gene start codon and/or one or morestop codons positioned downstream of the gene start codon and upstreamof the gene stop codon.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity and a knockout mutated ptr gene comprising amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon.

In one embodiment the present invention provides a cell comprising amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, a knockout mutated ptr gene comprising amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon and a knockout mutated Tsp gene comprising a mutation to thegene start codon and/or one or more stop codons positioned downstream ofthe gene start codon and upstream of the gene stop codon.

The cell provided by the second aspect of the present inventionovercomes the above described disadvantages of knockout mutation methodsemploying DNA insertion typically used in the art to provide proteasedeficient strains. In the present invention the knockout mutations tothe ptr gene and/or the Tsp gene are provided by a mutation to the genestart codon and/or one or more stop codons positioned downstream of thegene start codon and upstream of the gene stop codon. A mutation, suchas a missense point mutation, to the target knockout gene start codonensures that the target gene does not comprise a suitable start codon atthe start of the coding sequence. The insertion of one or more stopcodons positioned between the gene start codon and stop codon ensuresthat even if transcription of the gene is initiated, the full codingsequence will not be transcribed. The host genome required minimaldisruption to mutate the start codon and/or insert one or more stopcodons, thereby minimizing the deleterious effects of genome disruptionon the expression of the target protein and/or growth of the host cell.The cell of the present invention may also be more suitable forproduction of proteins intended for use as therapeutics due to theminimal disruption to the cell genome.

In a third aspect, the present invention provides a method for producinga recombinant protein of interest comprising expressing the recombinantprotein of interest in a recombinant gram-negative bacterial cell asdefined above in the first aspect or second aspect of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows the 5′ end of the wild type ptr (protease III) (SEQ ID NO:21) and knockout mutated ptr (protease III) (SEQ ID NO: 22) protein andgene sequences.

FIG. 1b shows the 5′ end of the wild type Tsp (SEQ ID NO: 23) andknockout mutated Tsp (SEQ ID NO: 24) protein and gene sequences.

FIG. 1c shows a region of the wild type DegP (SEQ ID NO: 25) and mutatedDegP (SEQ ID NO: 26) protein and gene sequences.

FIG. 2 shows the growth of E. coli strain MXE001 carrying a knockoutmutated Tsp gene and E. coli strain MXE005 carrying a knockout mutatedTsp gene and mutated DegP gene compared to E. coli wild type W3110.

FIG. 3 shows the expression of a Fab′ in MXE005 and MXE001 compared towild type W3110.

FIG. 4 shows the growth of E. coli strain MXE004 carrying a knockoutmutated Tsp gene and a knockout mutated protease III compared to wildtype W3110.

FIG. 5 shows the expression of a Fab′ in MXE004 and W3110.

FIG. 6 shows the expression of a Fab in MXE001, MXE004, MXE005 andW3110.

FIG. 7 shows the light chain (L chain), heavy chain (H chain) and Fab′expression during a fermentation experiment for MXE001, MXE005 and wildtype W3110.

FIG. 8 shows the results of a western blot analysis for wild type W3110,MXE001 and MXE005 showing relative fragmentation of a Fab′.

FIG. 9 shows the growth profile of MXE001 compared to control W3110.

FIG. 10 shows Fab′ yield from the supernatant (dotted lines) andperiplasm (solid lines) from E. coli strain MXE001 compared to controlE. coli W3110.

FIG. 11 shows the total Fab′ yield from the supernatant and periplasm ofthe E. coli strain MXE001 compared to control W3110.

FIG. 12 shows the Fab′ specific production rate of E. coli strain MXE001compared to the control W3110.

FIG. 13 shows the growth profile of MXE004 and MXE005 compared tocontrol W3110.

FIG. 14 shows Fab′ yields from the supernatant (dotted lines) andperiplasm (solid lines) of E. coli strains MXE004, MXE005 and the W3110control.

FIG. 15 shows the total Fab′ yield from the supernatant and periplasm ofthe E. coli strains MXE004 and MXE005.

FIG. 16 shows the Fab′ specific production rate of E. coli strainsMXE004 and MXE005 and the W3110 control.

FIG. 17 shows the growth profile of E. coli strains W3110, MXE001,MXE004 and MXE005 compared to E. coli strains XL1 Blue, TOP10, Stbl 3and Sure.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the DNA sequence of the non-mutated Tsp gene includingthe 6 nucleotides ATGAAC upstream of the start codon.

SEQ ID NO:2 is the amino acid sequence of the non-mutated Tsp protein.

SEQ ID NO:3 is the DNA sequence of a mutated knockout Tsp gene includingthe 6 nucleotides ATGAAT upstream of the start codon.

SEQ ID NO:4 is the DNA sequence of the non-mutated Protease III gene.

SEQ ID NO:5 is the amino acid sequence of the non-mutated Protease IIIprotein.

SEQ ID NO:6 is the DNA sequence of a mutated knockout Protease III gene.

SEQ ID NO:7 is the DNA sequence of the non-mutated DegP gene.

SEQ ID NO:8 is the amino acid sequence of the non-mutated DegP protein.

SEQ ID NO:9 is the DNA sequence of a mutated DegP gene.

SEQ ID NO:10 is the amino acid sequence of a mutated DegP protein.

SEQ ID NO:11 is the amino acid sequence of the light chain variableregion of an anti-TNF antibody.

SEQ ID NO:12 is the amino acid sequence of the heavy chain variableregion of an anti-TNF antibody.

SEQ ID NO:13 is the amino acid sequence of the light chain of ananti-TNF antibody.

SEQ ID NO:14 is the amino acid sequence of the heavy chain of ananti-TNF antibody.

SEQ ID NO:15 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO:16 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO:17 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO:18 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO:19 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

SEQ ID NO:20 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In the first aspect and second aspect of the present invention thepresent inventors have provided a recombinant gram-negative bacterialcell suitable for expressing a protein of interest which comprises onlythe minimal mutations to the genome required to introduce one or moreprotease mutations. In the first aspect of the invention, the bacterialcell only differs from a wild-type bacterial cell by the one or moremutated protease genes selected from a mutated DegP gene encoding a DegPprotein having chaperone activity and reduced protease activity; amutated ptr; and a mutated Tsp gene and optionally a polynucleotidesequence encoding a protein of interest. In the second aspect of thepresent invention the bacterial cell comprises knockout mutations of Tspand/or Protease III, wherein the Tsp and/or Protease III gene comprisesa mutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon.

The cells provided by the first and second aspects of the presentinvention have reduced protease activity compared to non-mutated cell,which may reduce proteolysis of a recombinant protein of interest,particularly proteins of interest which are proteolytically sensitive.Therefore, one or more of the gram-negative cells provided by the firstand second aspects of the present invention may provide higher yield ofthe intact recombinant protein of interest and a lower yield, orpreferably no yield, of proteolytic fragments of the protein of interestcompared to a non-mutated bacterial cell.

The skilled person would easily be able to test a candidate cell cloneto see if it has the desired yield of a protein of interest usingmethods well known in the art including a fermentation method, ELISA andprotein G hplc. Suitable fermentation methods are described in HumphreysD P, et al. (1997). Formation of dimeric Fabs in E. coli: effect ofhinge size and isotype, presence of interchain disulphide bond, Fab′expression levels, tail piece sequences and growth conditions. J.IMMUNOL. METH. 209: 193-202; Backlund E. Reeks D. Markland K. Weir N.Bowering L. Larsson G. Fedbatch design for periplasmic product retentionin Escherichia coli, Journal Article. Research Support, Non-U.S. Gov'tJournal of Biotechnology. 135(4):358-65, 2008 Jul. 31; Champion K M.Nishihara J C. Joly J C. Arnott D. Similarity of the Escherichia coliproteome upon completion of different biopharmaceutical fermentationprocesses. [Journal Article] Proteomics. 1(9):1133-48, 2001 September;and Horn U. Strittmatter W. Krebber A. Knupfer U. Kujau M. Wenderoth R.Muller K. Matzku S. Pluckthun A. Riesenberg D. High volumetric yields offunctional dimeric miniantibodies in Escherichia coli, using anoptimized expression vector and high-cell-density fermentation undernon-limited growth conditions, Journal Article. Research Support,Non-U.S. Gov't Applied Microbiology & Biotechnology. 46(5-6):524-32,1996 December. The skilled person would also easily be able to testsecreted protein to see if the protein is correctly folded using methodswell known in the art, such as protein G HPLC, circular dichroism, NMR,X-Ray crystallography and epitope affinity measurement methods.

One or more of the recombinant bacterial cells of the first and secondaspects of the present invention may exhibit significantly improvedprotein yield compared to a non-mutated bacterial cell. The improvedprotein yield may be the periplasmic protein yield and/or thesupernatant protein yield. One or more of the recombinant bacterialcells of the first and second aspects of the present invention may becapable of faster rate of production of a protein of interest and,therefore, the same quantity of a protein of interest may be produced ina shorter time compared to a non-mutated bacterial cell. The faster rateof production of a protein of interest may be especially significantover the initial period of growth of the cell, for example over thefirst 5, 10, 20 or 30 hours post induction of protein expression.

The cells according to the present invention comprising the Tspmutation, which is preferably the knockout mutation, either alone or incombination with the DegP mutation or the Protease III mutation, areparticularly preferred. These cells exhibit a higher yield and a fasterinitial yield of a protein of interest compared to a non-mutated cell.Example of such cell lines comprising the mutated Tsp gene either aloneor in combination with mutated DegP gene or the mutated ptr gene aremutant E. coli cell strains MXE001 having genotype ΔTsp and deposited on21 May 2009 at the National Collection of Type Cultures, HPA, UnitedKingdom, under Accession number NCTC13444, MXE004 having genotype ΔTspΔptr, and deposited on 21 May 2009 at the National Collection of TypeCultures, HPA, United Kingdom, under Accession number NCTC13447, andMXE005 having genotype ΔTsp, DegP S210A and deposited on 21 May 2009 atthe National Collection of Type Cultures, HPA, United Kingdom, underAccession number NCTC13448.

Further, one or more of the cells may show good growth characteristicsincluding cell growth and/or reproduction which may be substantially thesame as a non-mutated bacterial cell or improved compared to anon-mutated bacterial cell.

The genome of the cell according to the first aspect of the presentinvention has had minimal disruption to the genome compared to awild-type cell thereby reducing deleterious effects of other mutationstypically found in host cells on the expression of other cellularproteins. Accordingly, one or more of the recombinant host cellsaccording to the first aspect of the present invention may exhibitimproved protein expression and/or improved growth characteristicscompared to cells comprising further genetically engineered mutations tothe genomic sequence.

The genome of the cell according to the second aspect of the presentinvention has had minimal disruption to the genome to introduce theknockout mutations thereby reducing deleterious effects of creatingprotease gene knockouts by inserting DNA, such as antibiotic resistancemarkers. Accordingly, one or more of the recombinant host cellsaccording to the second aspect of the present invention may exhibitimproved protein expression and/or improved growth characteristicscompared to cells comprising protease knockout mutations created by theinsertion of DNA, such as antibiotic resistance markers.

The cells provided by the first and second aspects of the presentinvention are also more suitable for use to produce therapeutic proteinscompared to cells comprising further disruptions to the cell genome.

The present invention will now be described in more detail. Allembodiments herein described refer to the first, second and thirdaspects of the present invention unless specifically stated otherwise.

The terms “protein” and “polypeptide” are used interchangeably herein,unless the context indicates otherwise. “Peptide” is intended to referto 10 or less amino acids.

The terms “polynucleotide” includes a gene, DNA, cDNA, RNA, mRNA etcunless the context indicates otherwise.

As used herein, the term “comprising” in context of the presentspecification should be interpreted as “including”.

The non-mutated cell or control cell in the context of the presentinvention means a cell of the same type as the recombinant gram-negativecell of the invention wherein the cell has not been modified to carrythe above protease mutations. For example, a non-mutated cell may be awild-type cell and may be derived from the same population of host cellsas the cells of the invention before modification to introduce the oneor more mutations.

The expressions “cell”, “cell line”, “cell culture” and “strain” areused interchangeably.

The term “isogenic” in the context of the present invention means thatthe genome of the cell of the present invention has substantially thesame or the same genomic sequence compared to wild-type cell except forone or more of the above mutated protease genes and optionally apolynucleotide encoding a protein of interest. In this embodiment thecell according to the present invention comprises no furthernon-naturally occurring or genetically engineered mutations compared tothe wild-type cell. In one embodiment the cell according to the presentinvention may have substantially the same genomic sequence compared tothe wild-type cell except for the above protease mutations andoptionally a polynucleotide encoding a protein of interest taking intoaccount any naturally occurring mutations which may occur. It shouldalso be noted that during the introduction of the protease mutationsinto the strain, for example by a gene replacement vector, and duringthe introduction of the polynucleotide encoding the protein of interestinto the strain one or more further genomic mutations may be introducedinto the strain. Accordingly, in one embodiment the cell according tothe present invention may have substantially the same genomic sequencecompared to the wild-type cell except for the above protease mutationsand optionally a polynucleotide encoding a protein of interest takinginto account any naturally occurring mutations which may occur and anyfurther genomic mutations which may result from the introduction of theprotease mutations and/or the polynucleotide encoding the protein ofinterest.

Examples of gene mutations involved in cell metabolism and DNAreplication, which are commonly used in E. coli strains in the art butare not used in the cell according to the present invention includephoA, fhuA, lac, rec, gal, ara, arg, thi and pro.

In one embodiment, the cell according to the present invention may haveexactly the same genomic sequence compared to the wild-type cell exceptfor the above protease mutations and optionally a polynucleotideencoding a protein of interest.

The term “wild-type” in the context of the present invention means astrain of a gram-negative bacterial cell as it may occur in nature ormay be isolated from the environment, which does not carry anygenetically engineered mutations. An example of a wild-type strain of E.coli is W3110, such as W3110 K-12 strain. Examples of wild-type strainsinclude strains of the K-12 strain family which includes W3110 (F⁻ λ⁻rph-1 INV(rrnD, rrnE) ilvG) (ATCC27325), MG1655 (F⁻ λ⁻ ilvG-rfb-50rph-1) (ATCC700926), W1485 (F+ λ⁻ rph-1 rpoS396) (ATCC12435), W3101 (F⁻λ⁻ ilvG-IN(rrnD-rrnE)1 rph-1 galT22) and BW30270 (F⁻ λ⁻ fnr+). Furtherexamples of wild-type E. coli strains include the W strain (ATCC9637)and the B strain (ATCC23226).

Any suitable gram-negative bacterium may be used as the parental cellfor producing the recombinant cell of the present invention. Suitablegram-negative bacterium include Salmonella typhimurium, Pseudomonasfluorescens, Erwinia carotovora, Shigella, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Acinetobacter baumanniiand E. coli. Preferably the parental cell is E. coli. Any suitablestrain of E. coli may be used in the present invention as the parentalcell. Examples of suitable E. coli strains include the K-12 strainfamily which comprises W3110 (F⁻ λ⁻ rph-1 INV(rrnD, rrnE) ilvG)(ATCC27325), MG1655 (F⁻ λ⁻ ilvG-rfb-50 rph-1) (ATCC700926), W1485 (F+ λ⁻rph-1 rpoS396) (ATCC12435), W3101 (F⁻ λ⁻ ilvG-IN(rrnD-rrnE)1 rph-1galT22) and BW30270 (F⁻ λ⁻ fnr+). Further suitable E. coli strainsinclude the W strain (ATCC9637) and the B strain (ATCC23226). Preferablya wild-type W3110 strain, such as K-12 W3110, is used.

The cell according to the first aspect of the present is isogenic to awild-type bacterial cell except for the one or more mutated proteasegenes and optionally a polynucleotide sequence encoding a protein ofinterest. The cell according to the second aspect of the presentinvention is preferably also isogenic to a wild-type bacterial cellexcept for the one or more mutated protease genes and optionally apolynucleotide sequence encoding a protein of interest.

In a preferred embodiment, the cell is isogenic to a wild-type E. colicell except for the above protease mutations and optionally apolynucleotide encoding a protein of interest. More preferably the cellaccording to the present invention is isogenic to an E. coli strainW3110 except for the above protease mutations and optionally apolynucleotide encoding a protein of interest. Examples of othersuitable wild-type E. coli cells which the cell according to the presentinvention may be isogenic to except for the above protease mutations andoptionally the polynucleotide encoding a protein of interest are strainsof the K-12 strain family which includes W3110 (F⁻ λ⁻ rph-1 INV(rrnD,rrnE) ilvG) (ATCC27325), MG1655 (F⁻ λ⁻ ilvG-rfb-50 rph-1) (ATCC700926),W1485 (F+ λ⁻ rph-1 rpoS396) (ATCC12435), W3101 (F⁻ λ⁻ilvG-IN(rrnD-rrnE)1 rph-1 galT22) and BW30270 (F⁻ λ⁻ fnr+). Furthersuitable wild-type E. coli strains which the cell according to thepresent invention may be isogenic to except for the above proteasemutations and optionally the polynucleotide encoding a protein ofinterest are include the W strain (ATCC9637) and the B strain(ATCC23226).

The cell of the present invention may further differ from a wild-typecell by comprising a polynucleotide encoding the protein of interest. Inthis embodiment, the polynucleotide encoding the protein of interest maybe contained within a suitable expression vector transformed into thecell and/or integrated into the host cell's genome. In the embodimentwhere the polynucleotide encoding the protein of interest is insertedinto the host's genome, the cell of the present invention will alsodiffer from a wild-type cell due to the inserted polynucleotide sequenceencoding the protein of interest. Preferably the polynucleotide is in anexpression vector in the cell thereby causing minimal disruption to thehost cell's genome.

In certain embodiments of the present invention the recombinantgram-negative bacterial cell comprises a mutated DegP gene encoding aDegP protein having chaperone activity and reduced protease activity. Asused herein, “DegP” means a gene encoding DegP protein (also known asHtrA), which has dual function as a chaperone and a protease (Familiesof serine peptidases; Rawlings N D, Barrett A J. Methods Enzymol. 1994;244:19-61). The sequence of the non-mutated DegP gene is shown in SEQ IDNO: 7 and the sequence of the non-mutated DegP protein is shown in SEQID NO: 8.

At low temperatures DegP functions as a chaperone and at hightemperatures DegP has a preference to function as a protease (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and The proteolytic activity of the HtrA(DegP) protein from Escherichia coli at low temperatures, Skorko-GlonekJ et al Microbiology 2008, 154, 3649-3658).

In the embodiments where the cell comprises the DegP mutation the DegPmutation in the cell provides a mutated DegP gene encoding a DegPprotein having chaperone activity but not full protease activity.

The expression “having chaperone activity” in the context of the presentinvention means that the mutated DegP protein has the same orsubstantially the same chaperone activity compared to the wild-typenon-mutated DegP protein. Preferably, the mutated DegP gene encodes aDegP protein having 50% or more, 60% or more, 70% or more, 80% or more,90% or more or 95% or more of the chaperone activity of a wild-typenon-mutated DegP protein. More preferably, the mutated DegP gene encodesa DegP protein having the same chaperone activity compared to wild-typeDegP.

The expression “having reduced protease activity” in the context of thepresent invention means that the mutated DegP protein does not have thefull protease activity compared to the wild-type non-mutated DegPprotein. Preferably, the mutated DegP gene encodes a DegP protein having50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% orless of the protease activity of a wild-type non-mutated DegP protein.More preferably, the mutated DegP gene encodes a DegP protein having noprotease activity. The cell is not deficient in chromosomal DegP i.e.the DegP gene sequences has not been deleted or mutated to preventexpression of any form of DegP protein.

Any suitable mutation may be introduced into the DegP gene in order toproduce a protein having chaperone activity and reduced proteaseactivity. The protease and chaperone activity of a DegP proteinexpressed from a gram-negative bacterium may be easily tested by aperson skilled in the art by any suitable method such as the methoddescribed in Spiess et al wherein the protease and chaperone activitiesof DegP were tested on MalS, a natural substrate of DegP (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and also the method described in Theproteolytic activity of the HtrA (DegP) protein from Escherichia coli atlow temperatures, Skorko-Glonek J et at Microbiology 2008, 154,3649-3658.

DegP is a serine protease and has an active center consisting of acatalytic triad of amino acid residues of His105, Asp135 and Ser210(Families of serine peptidases, Methods Enzymol., 1994, 244:19-61Rawlings N and Barrett A). The DegP mutation to produce a protein havingchaperone activity and reduced protease activity may comprise amutation, such as a missense mutation to one, two or three of His105,Asp135 and Ser210. Accordingly, the mutated DegP gene may comprise:

-   -   a mutation to His105; or    -   a mutation to Asp135; or    -   a mutation to Ser210; or    -   a mutation to His105 and Asp135; or    -   a mutation to His105 and Ser210; or    -   a mutation to Asp135 and Ser210; or    -   a mutation to His105, Asp135 and Ser210.

One, two or three of His105, Asp135 and Ser210 may be mutated to anysuitable amino acid which results in a protein having chaperone activityand reduced protease activity. For example, one, two or three of His105,Asp135 and Ser210 may be mutated to a small amino acid such as Gly orAla. A further suitable mutation is to change one, two or three ofHis105, Asp135 and Ser210 to an amino acid having opposite propertiessuch as Asp135 being mutated to Lys or Arg, polar His105 being mutatedto a non-polar amino acid such as Gly, Ala, Val or Leu and smallhydrophilic Ser210 being mutated to a large or hydrophobic residue suchas Val, Leu, Phe or Tyr. Preferably, the DegP gene comprises the pointmutation S210A, as shown in FIG. 1c , which has been found to produce aprotein having chaperone activity but not protease activity (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M).

The present invention also provides a recombinant gram-negativebacterial cell comprising a mutated DegP gene encoding a DegP proteinhaving chaperone activity and reduced protease activity, wherein theDegP gene comprises a mutation to His105; or a mutation to Asp135; or amutation to His105 and Asp135; or a mutation to His105 and Ser210; or amutation to Asp135 and Ser210; or a mutation to His105, Asp135 andSer210, as discussed above.

DegP has two PDZ domains, PDZ1 (residues 260-358) and PDZ2 (residues359-448), which mediate protein-protein interaction (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M). In one embodiment of the present inventionthe degP gene is mutated to delete PDZ1 domain and/or PDZ2 domain. Thedeletion of PDZ1 and PDZ2 results in complete loss of protease activityof the DegP protein and lowered chaperone activity compared to wild-typeDegP protein whilst deletion of either PDZ1 or PDZ2 results in 5%protease activity and similar chaperone activity compared to wild-typeDegP protein (A Temperature-Dependent Switch from Chaperone to Proteasein a Widely Conserved Heat Shock Protein. Cell, Volume 97, Issue 3,Pages 339-347. Spiess C, Beil A, Ehrmann M).

The present invention also provides a recombinant gram-negativebacterial cell comprising a mutated DegP gene encoding a DegP proteinhaving chaperone activity and reduced protease activity, wherein thedegP gene is mutated to delete PDZ1 domain and/or PDZ2 domain, asdiscussed above.

The mutated DegP gene may also comprise a silent non-naturally occurringrestriction site, such as Ase I in order to aid in identification andscreening methods, for example as shown in FIG. 1 c.

The preferred sequence of the mutated DegP gene comprising the pointmutation S210A and an Ase I restriction marker site is provided in SEQID NO: 9 and the encoded protein sequence is shown in SEQ ID NO: 10. Themutations which have been made in the mutated DegP sequence of SEQ IDNO: 9 are shown in FIG. 1 c.

In the embodiments of the present invention wherein the cell comprises amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, one or more of the cells provided by thepresent invention may provide improved yield of correctly foldedproteins from the cell relative to mutated cells wherein the DegP genehas been mutated to knockout DegP preventing DegP expression, such aschromosomal deficient DegP. In a cell comprising a knockout mutated DegPgene preventing DegP expression, the chaperone activity of DegP is lostcompletely whereas in the cell according to the present invention thechaperone activity of DegP is retained whilst the full protease activityis lost. In these embodiments, one or more cells according to thepresent invention have a lower protease activity to prevent proteolysisof the protein whilst maintaining the chaperone activity to allowcorrect folding and transportation of the protein in the host cell.

The skilled person would easily be able to test secreted protein to seeif the protein is correctly folded using methods well known in the art,such as protein G HPLC, circular dichroism, NMR, X-Ray crystallographyand epitope affinity measurement methods.

In these embodiments, one or more cells according to the presentinvention may have improved cell growth compared to cells carrying amutated knockout DegP gene preventing DegP expression. Without wishingto be bound by theory improved cell growth may be exhibited due to theDegP protease retaining chaperone activity which may increase capacityof the cell to process all proteins which require chaperone activity.Accordingly, the production of correctly folded proteins necessary forthe cell's growth and reproduction may be increased in one or more ofthe cells of the present invention compared to cells carrying a DegPknockout mutation thereby improving the cellular pathways regulatinggrowth. Further, known DegP protease deficient strains are generallytemperature-sensitive and do not typically grow at temperatures higherthan about 28° C. However, the cells according to the present inventionare not temperature-sensitive and may be grown at temperatures of 28° C.or higher, including temperatures of approximately 30° C. toapproximately 37° C., which are typically used for industrial scaleproduction of proteins from bacteria.

In certain embodiments of the present invention the recombinantgram-negative bacterial cell comprises a knockout mutated ptr gene. Asused herein, “ptr gene” means a gene encoding Protease III, a proteasewhich degrades high molecular weight proteins. The sequence of thenon-mutated ptr gene is shown in SEQ ID NO: 4 and the sequence of thenon-mutated Protease III protein is shown in SEQ ID NO: 5.

In certain embodiments of the present invention the recombinantgram-negative bacterial cell comprises a knockout mutated Tsp gene. Asused herein, “Tsp gene” means a gene encoding protease Tsp (also knownas Prc) which is a periplasmic protease capable of acting onPenicillin-binding protein-3 (PBP3) and phage tail proteins. Thesequence of the non-mutated Tsp gene is show in SEQ ID NO: 1 and thesequence of the non-mutated Tsp protein is shown in SEQ ID NO: 2.

In the first aspect of the present invention, reference to the mutatedptr gene or mutated ptr gene encoding Protease III, refers to either amutated ptr gene encoding a Protease III protein having reduced proteaseactivity or a knockout mutated ptr gene, unless otherwise indicated.

In the first aspect of the present invention, reference to the mutatedTsp gene or mutated Tsp gene encoding Tsp, refers to either a mutatedTsp gene encoding a Tsp protein having reduced protease activity or aknockout mutated Tsp gene, unless otherwise indicated.

In the first aspect of the present invention the expressions “mutatedptr gene encoding a Protease III protein having reduced proteaseactivity” and “mutated Tsp gene encoding a Tsp protein having reducedprotease activity” in the context of the present invention means thatthe mutated ptr gene or the mutated Tsp gene does not have the fullprotease activity compared to the wild-type non-mutated ptr gene or Tspgene.

In the first aspect of the present invention, preferably, the mutatedptr gene encodes a Protease III having 50% or less, 40% or less, 30% orless, 20% or less, 10% or less or 5% or less of the protease activity ofa wild-type non-mutated Protease III protein. More preferably, themutated ptr gene encodes a Protease III protein having no proteaseactivity. In this embodiment the cell is not deficient in chromosomalptr i.e. the ptr gene sequence has not been deleted or mutated toprevent expression of any form of Protease III protein.

Any suitable mutation may be introduced into the ptr gene in order toproduce a Protease III protein having reduced protease activity. Theprotease activity of a Protease III protein expressed from agram-negative bacterium may be easily tested by a person skilled in theart by any suitable method in the art.

In the first aspect of the present invention, preferably, the mutatedTsp gene encodes a Tsp protein having 50% or less, 40% or less, 30% orless, 20% or less, 10% or less or 5% or less of the protease activity ofa wild-type non-mutated Tsp protein. More preferably, the mutated Tspgene encodes a Tsp protein having no protease activity. In thisembodiment the cell is not deficient in chromosomal Tsp i.e. the Tspgene sequence has not been deleted or mutated to prevent expression ofany form of Tsp protein.

Any suitable mutation may be introduced into the Tsp gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a Tsp protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Keiler et al (Identificationof Active Site Residues of the Tsp Protease* THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 270, No. 48, Issue of December 1, pp. 28864-28868, 1995Kenneth C. Keiler and Robert T. Sauer) wherein the protease activitiesof Tsp was tested.

Tsp has been reported in Keiler et al (supra) as having an active sitecomprising residues S430, D441 and K455 and residues G375, G376, E433and T452 are important for maintaining the structure of Tsp. Keiler etal (supra) reports findings that the mutated Tsp genes S430A, D441A,K455A, K455H, K455R, G375A, G376A, E433A and T452A had no detectableprotease activity. It is further reported that the mutated Tsp geneS430C displayed about 5-10% wild-type activity. Accordingly, the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one or more ofresidues S430, D441, K455, G375, G376, E433 and T452. Preferably the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one, two or allthree of the active site residues S430, D441 and K455.

According the mutated Tsp gene may comprise:

-   -   a mutation to 5430; or    -   a mutation to D441; or    -   a mutation to K455; or    -   a mutation to 5430 and D441; or    -   a mutation to 5430 and K455; or    -   a mutation to D441 and K455; or    -   a mutation to 5430, D441 and K455.

One or more of 5430, D441, K455, G375, G376, E433 and T452 may bemutated to any suitable amino acid which results in a protein havingreduced protease activity. Examples of suitable mutations are S430A,S430C, D441A, K455A, K455H, K455R, G375A, G376A, E433A and T452A. Themutated Tsp gene may comprise one, two or three mutations to the activesite residues, for example the gene may comprise:

-   -   S430A or S430C; and/or    -   D441A; and/or    -   K455A or K455H or K455R.

Preferably, the Tsp gene comprises the point mutation S430A or S430C.

The present invention also provides a recombinant gram-negativebacterial cell comprising a mutated Tsp gene, wherein the mutated Tspgene encodes a Tsp protein having reduced protease activity, wherein theTsp gene comprise a mutation, such as a missense mutation to one or moreof residues S430, D441, K455, G375, G376, E433 and T452, as discussedabove.

In the first aspect of the present invention the expression “knockoutmutated ptr gene” and “knockout mutated Tsp gene” in the context of thepresent invention means that the gene comprises one or more mutationsthereby causing no expression of the protein encoded by the gene toprovide a cell deficient in the protein encoded by the knockout mutatedgene. The knockout gene may be partially or completely transcribed butnot translated into the encoded protein.

In the first aspect of the present invention, the knockout mutated ptrgene and/or knockout mutated Tsp gene may be mutated in any suitableway, for example by one or more deletion, insertion, point, missense,nonsense and frameshift mutations, to cause no expression of theprotein. For example, the gene may be knocked out by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence.

In a preferred embodiment of the first aspect of the present inventionthe gene is not mutated by insertion of a foreign DNA sequence, such asan antibiotic resistance marker, into the gene coding sequence.Preferably the Tsp gene and/or Protease III gene comprise a mutation tothe gene start codon and/or one or more stop codons positioneddownstream of the gene start codon and upstream of the gene stop codonthereby preventing expression of the Tsp protein and/or Protease IIIprotein.

The cell according to the second aspect of the present inventioncomprises Tsp and/or Protease III knockout mutations where the Tsp geneand/or Protease III gene comprise a mutation to the gene start codonand/or one or more stop codons positioned downstream of the gene startcodon and upstream of the gene stop codon thereby preventing expressionof the Tsp protein and/or Protease III protein.

A mutation to the target knockout gene start codon causes loss offunction of the start codon and thereby ensures that the target genedoes not comprise a suitable start codon at the start of the codingsequence. The mutation to the start codon may be a missense mutation ofone, two or all three of the nucleotides of the start codon.Alternatively or additionally the start codon may be mutated by aninsertion or deletion frameshift mutation.

The ptr gene and Tsp gene each comprise an ATG start codon. If the genecomprises more than one suitably positioned start codon, as found in theTsp gene where two ATG codons are present at the 5′ end of the codingsequence, one or both of the ATG codons may be mutated by a missensemutation.

In a preferred embodiment the ptr gene is mutated to change the ATGstart codon to ATT, as shown in FIG. 1a . In a preferred embodiment theTsp gene is mutated at the second ATG codon (codon 3) to TCG, as shownin FIG. 1 b.

The knockout mutated ptr gene and/or the knockout mutated Tsp gene mayalternatively or additionally comprise one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon. Preferably the knockout mutated ptr gene and/or the knockoutmutated Tsp gene comprise both a missense mutation to the start codonand one or more inserted stop codons.

The one or more inserted stop codons are preferably in-frame stopcodons. However the one or more inserted stop codons may alternativelyor additionally be out-of-frame stop codons. One or more out-of-framestop codons may be required to stop translation where an out-of-framestart codon is changed to an in-frame start codon by an insertion ordeletion frameshift mutation. The one or more stop codons may beintroduced by any suitable mutation including a nonsense point mutationand a frameshift mutation. The one or more stop codons are preferablyintroduced by a frameshift mutation and/or an insertion mutation,preferably by replacement of a segment of the gene sequence with asequence comprising a stop codon. For example an Ase I restriction sitemay be inserted, which comprises the stop codon TAA.

In a preferred embodiment the ptr gene is mutated to insert an in-framestop codon by insertion of an Ase I restriction site, as shown in FIG. 1a.

In a preferred embodiment the Tsp gene is mutated to delete “T” from thefifth codon thereby causing a frameshift resulting in stop codons atcodons 11 and 16, as shown in FIG. 1b . In a preferred embodiment theTsp gene is mutated to insert an Ase I restriction site to create athird in-frame stop codon at codon 21, as shown in FIG. 1 b.

In a preferred embodiment the knockout mutated ptr gene has the DNAsequence of SEQ ID NO: 6. The mutations which have been made in theknockout mutated ptr gene sequence of SEQ ID NO: 6 are shown in FIG. 1a.

In a preferred embodiment the knockout mutated Tsp gene has the DNAsequence of SEQ ID NO: 3, which includes the 6 nucleotides ATGAATupstream of the start codon. The mutations which have been made in theknockout mutated Tsp sequence of SEQ ID NO: 3 are shown in FIG. 1b . Inone embodiment the mutated Tsp gene has the DNA sequence of nucleotides7 to 2048 of SEQ ID NO: 3.

The above described knockout mutations are advantageous because theycause minimal or no disruption to the chromosomal DNA upstream ordownstream of the target knockout gene site and do not require theinsertion and retention of foreign DNA, such as antibiotic resistancemarkers, which may affect the cell's suitability for expressing aprotein of interest, particularly therapeutic proteins. Accordingly, oneor more of the cells according to the present invention may exhibitimproved growth characteristics and/or protein expression compared tocells wherein the protease gene has been knocked out by insertion offoreign DNA into the gene coding sequence.

Many genetically engineered mutations including knockout mutationsinvolve the use of antibiotic resistance markers which allow theselection and identification of successfully mutated cells. However, asdiscussed above, there are a number of disadvantages to using antibioticresistance markers.

A further embodiment of the present invention overcomes the abovedisadvantages of using antibiotic resistance markers wherein the mutatedprotease genes selected from one or more of a mutated DegP gene encodinga DegP protein having chaperone activity but not protease activity; amutated ptr gene encoding Protease III; and a mutated Tsp gene encodingprotease Tsp, are mutated to comprise one or more restriction markersites. The restriction sites are genetically engineered into the geneand are non-naturally occurring. The restriction marker sites areadvantageous because they allow screening and identification ofcorrectly modified cells which comprise the required chromosomalmutations. Cells which have been modified to carry one or more of themutated protease genes may be analyzed by PCR of genomic DNA from celllysates using oligonucleotide pairs designed to amplify a region of thegenomic DNA comprising a non-naturally occurring restriction markersite. The amplified DNA may then be analyzed by agarose gelelectrophoresis before and after incubation with a suitable restrictionenzyme capable of digesting the DNA at the non-naturally occurringrestriction marker site. The presence of DNA fragments after incubationwith the restriction enzyme confirms that the cells have beensuccessfully modified to carry the one or more mutated protease genes.

In the embodiment wherein the knockout mutated ptr gene has the DNAsequence of SEQ ID NO: 6, the oligonucleotide primer sequences shown inSEQ ID NO: 17 and SEQ ID NO: 18 may be used to amplify the region of theDNA comprising the non-naturally occurring Ase I restriction site fromthe genomic DNA of transformed cells. The amplified genomic DNA may thenbe incubated with Ase I restriction enzyme and analyzed by gelelectrophoresis to confirm the presence of the mutated ptr gene in thegenomic DNA.

In the embodiment wherein the knockout mutated Tsp gene has the DNAsequence of SEQ ID NO: 3 or nucleotides 7 to 2048 of SEQ ID NO:3, theoligonucleotide primer sequences shown in SEQ ID NO: 15 and SEQ ID NO:16may be used to amplify the region of the DNA comprising thenon-naturally occurring Ase I restriction site from the genomic DNA oftransformed cells. The amplified genomic DNA may then be incubated withAse I restriction enzyme and analyzed by gel electrophoresis to confirmthe presence of the mutated Tsp gene in the genomic DNA.

In the embodiment wherein the mutated DegP gene has the DNA sequence ofSEQ ID NO: 9, the oligonucleotide primer sequences shown in SEQ ID NO:19 and SEQ ID NO: 20 may be used to amplify the region of the DNAcomprising the non-naturally occurring Ase I restriction site from thegenomic DNA of transformed cells. The amplified genomic DNA may then beincubated with Ase I restriction enzyme and analyzed by gelelectrophoresis to confirm the presence of the mutated DegP gene in thegenomic DNA.

The one or more restriction sites may be introduced by any suitablemutation including by one or more deletion, insertion, point, missense,nonsense and frameshift mutations. A restriction site may be introducedby the mutation of the start codon and/or mutation to introduce the oneor more stop codons, as described above. This embodiment is advantageousbecause the restriction marker site is a direct and unique marker of theknockout mutations introduced.

A restriction maker site may be inserted which comprises an in-framestop codon, such as an Ase I restriction site. This is particularlyadvantageous because the inserted restriction site serves as both arestriction marker site and a stop codon to prevent full transcriptionof the gene coding sequence. For example, in the embodiment wherein astop codon is introduced to the ptr gene by introduction of an Ase Isite, this also creates a restriction site, as shown in FIG. 1a . Forexample, in the embodiment wherein a stop codon is introduced to the Tspgene at codon 21 by introduction of an Ase I site, this also creates arestriction site, as shown in FIG. 1 b.

A restriction marker site may be inserted by the mutation to the startcodon and optionally one or more further point mutations. In thisembodiment the restriction marker site is preferably an EcoR Irestriction site. This is particularly advantageous because the mutationto the start codon also creates a restriction marker site. For example,in the embodiment wherein the start codon of the ptr gene is changed toATT, this creates an EcoR I marker site, as shown in FIG. 1a . Forexample, in the embodiment wherein the start codon (codon 3) of the Tspgene is changed from ATG to TCG, as shown in FIG. 1b , a further pointmutation of codon 2 from AAC to AAT and mutation of codon 3 ATG to TCGcreates an EcoR I restriction marker site, as shown in FIG. 1 b.

In the DegP gene, a marker restriction site may be introduced usingsilent codon changes. For example, an Ase I site may be used as a silentrestriction marker site, wherein the TAA stop codon is out-of-frame, asshown in FIG. 1 c.

In the embodiments of the present invention, wherein the ptr gene and/orthe Tsp gene are mutated to encode a Protease III or Tsp having reducedprotease activity, one or more marker restriction site may be introducedusing silent codon changes.

The recombinant gram-negative bacterial cell according to the presentinvention may be produced by any suitable means. The skilled personknows of suitable techniques which may be used to replace a chromosomalgene sequence with a mutated gene sequence. Suitable vectors may beemployed which allow integration into the host chromosome by homologousrecombination.

Suitable gene replacement methods are described, for example, inHamilton et at (New Method for Generating Deletions and GeneReplacements in Escherichia coli, Hamilton C. M. et al., Journal ofBacteriology September 1989, Vol. 171, No. 9 p 4617-4622), Skorupski etat (Positive selection vectors for allelic exchange, Skorupski K andTaylor R. K., Gene, 1996, 169, 47-52), Kiel et at (A general method forthe construction of Escherichia coli mutants by homologous recombinationand plasmid segregation, Kiel J. A. K. W. et al, Mol Gen Genet 1987,207:294-301), Blomfield et at (Allelic exchange in Escherichia coliusing the Bacillus subtilis sacB gene and a temperature sensitive pSC101replicon, Blomfield I. C. et al., Molecular Microbiology 1991, 5(6),1447-1457) and Ried et al. (An nptI-sacB-sacR cartridge for constructingdirected, unmarked mutations in Gram-negative bacteria by markerexchange-eviction mutagenesis, Ried J. L. and Collmer A., Gene 57 (1987)239-246). A suitable plasmid which enables homologousrecombination/replacement is the pKO3 plasmid (Link et al., 1997,Journal of Bacteriology, 179, 6228-6237).

Successfully mutated strains may be identified using methods well knownin the art including colony PCR DNA sequencing and colony PCRrestriction enzyme mapping.

In the embodiment wherein the cell comprises two or three of the mutatedprotease genes, the mutated protease may be introduced into thegram-negative bacterium on the same or different vectors.

In one embodiment the present invention provides a mutant E. coli cellstrain MXE001 having genotype ΔTsp and deposited on 21 May 2009 at theNational Collection of Type Cultures, HPA, United Kingdom, underAccession number NCTC13444.

In a further embodiment the present invention provides a mutant E. colicell strain MXE002 having genotype Δptr and deposited on 21 May 2009 atthe National Collection of Type Cultures, HPA, United Kingdom, underAccession number NCTC13445.

In one embodiment the present invention provides a mutant E. coli cellstrain MXE003 having genotype DegP S210A and deposited on 21 May 2009 atthe National Collection of Type Cultures, HPA, United Kingdom, underAccession number NCTC13446.

In a further embodiment the present invention provides a mutant E. colicell strain MXE004 having genotype ΔTsp Δptr, and deposited on 21 May2009 at the National Collection of Type Cultures, HPA, United Kingdom,under Accession number NCTC13447.

In one embodiment the present invention provides a mutant E. coli cellstrain MXE005 having genotype ΔTsp, DegP S210A and deposited on 21 May2009 at the National Collection of Type Cultures, HPA, United Kingdom,under Accession number NCTC13448.

In a further embodiment the present invention provides a mutant E. colicell strain MXE006 having genotype Δptr, DegP S210A and deposited on 21May 2009 at the National Collection of Type Cultures, HPA, UnitedKingdom, under Accession number NCTC13449.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ompT gene, such asbeing deficient in chromosomal ompT. In one embodiment the cellaccording to the present invention does not carry any further knockoutmutated protease genes apart from the knockout mutated ptr gene and/orthe knockout mutated Tsp gene.

The cell according to the present invention may further comprise apolynucleotide sequence encoding a protein of interest. Thepolynucleotide sequence encoding the protein of interest may beexogenous or endogenous. The polynucleotide sequence encoding theprotein of interest may be integrated into the host's chromosome or maybe non-integrated in a vector, typically a plasmid.

In one embodiment the cell according to the present invention expressesa protein of interest. “Protein of interest” in the context of thepresent specification is intended to refer to polypeptide forexpression, usually a recombinant polypeptide. However, the protein ofinterest may be an endogenous protein expressed from an endogenous genein the host cell.

As used herein, a “recombinant polypeptide” refers to a protein that isconstructed or produced using recombinant DNA technology. The protein ofinterest may be an exogenous sequence identical to the endogenousprotein or a mutated version thereof, for example with attenuatedbiological activity, or fragment thereof, expressed from an exogenousvector. Alternatively, the protein of interest may be a heterologousprotein, not normally expressed by the host cell.

The protein of interest may be any suitable protein including atherapeutic, prophylactic or diagnostic protein.

In one embodiment the protein of interest is useful in the treatment ofdiseases or disorders including inflammatory diseases and disorders,immune disease and disorders, fibrotic disorders and cancers.

The term “inflammatory disease” or “disorder” and “immune disease ordisorder” includes rheumatoid arthritis, psoriatic arthritis, still'sdisease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerativecolitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis,atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetesmellitus, transplantation and graft-versus-host disease.

The term “fibrotic disorder” includes idiopathic pulmonary fibrosis(IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabeticnephropathy, IgA nephropathy, hypertension, end-stage renal disease,peritoneal fibrosis (continuous ambulatory peritoneal dialysis), livercirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiacreactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty,coronary bypass surgery, arthroplasty and cataract surgery.

The term “cancer” includes a malignant new growth that arises fromepithelium, found in skin or, more commonly, the lining of body organs,for example: breast, ovary, prostate, lung, kidney, pancreas, stomach,bladder or bowel. Cancers tend to infiltrate into adjacent tissue andspread (metastasise) to distant organs, for example: to bone, liver,lung or the brain.

The protein may be a proteolytically-sensitive polypeptide, i.e.proteins that are prone to be cleaved, susceptible to cleavage, orcleaved by one or more gram-negative bacterial, such as E. coli,proteases, either in the native state or during secretion. In oneembodiment the protein of interest is proteolytically-sensitive to aprotease selected from DegP, Protease III and Tsp. In one embodiment theprotein of interest is proteolytically-sensitive to the proteases DegPand Protease III. In one embodiment the protein of interest isproteolytically sensitive to the proteases DegP and Tsp. In oneembodiment the protein of interest is proteolytically-sensitive to theproteases Tsp and Protease III. In one embodiment the protein ofinterest is proteolytically sensitive to the proteases DegP, ProteaseIII and Tsp.

Preferably the protein is a eukaryotic polypeptide.

The protein of interest expressed by the cells according to theinvention may, for example be an immunogen, a fusion protein comprisingtwo heterologous proteins or an antibody. Antibodies for use as theprotein of interest include monoclonal, multi-valent, multi-specific,humanized, fully human or chimeric antibodies. The antibody can be fromany species but is preferably derived from a monoclonal antibody, ahuman antibody, or a humanized fragment. The antibody can be derivedfrom any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass ofimmunoglobulin molecule and may be obtained from any species includingfor example mouse, rat, shark, rabbit, pig, hamster, camel, llama, goator human. Parts of the antibody fragment may be obtained from more thanone species for example the antibody fragments may be chimeric. In oneexample the constant regions are from one species and the variableregions from another.

The antibody may be a complete antibody molecule having full lengthheavy and light chains or a fragment thereof, e.g. VH, VL, VHH, Fab,modified Fab, Fab′, F(ab′)₂, Fv, scFv fragment, Fab-Fv, or a dualspecificity antibody, such as a Fab-dAb, as described inPCT/GB2008/003331.

The antibody may be specific for any target antigen. The antigen may bea cell-associated protein, for example a cell surface protein on cellssuch as bacterial cells, yeast cells, T-cells, endothelial cells ortumour cells, or it may be a soluble protein. Antigens of interest mayalso be any medically relevant protein such as those proteinsupregulated during disease or infection, for example receptors and/ortheir corresponding ligands. Particular examples of cell surfaceproteins include adhesion molecules, for example integrins such as β1integrins e.g. VLA-4, E-selectin, P selectin or L-selectin, CD2, CD3,CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33,CD38, CD40, CD40L, CD45, CDW52, CD69, CD134 (OX40), ICOS, BCMP7, CD137,CD27L, CDCP1, CSF1 or CSF1-Receptor, DPCR1, DPCR1, dudulin2, FLJ20584,FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK,MAL2, MRP2, nectin-like2, NKCC1, PTK7, RAIG1, TCAM1, SC6, BCMP101,BCMP84, BCMP11, DTD, carcinoembryonic antigen (CEA), human milk fatglobulin (HMFG1 and 2), MHC Class I and MHC Class II antigens, KDR andVEGF, and where appropriate, receptors thereof.

Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-12, IL-13, IL-14, IL-16 or IL-17, such as IL17Aand/or IL17F, viral antigens for example respiratory syncytial virus orcytomegalovirus antigens, immunoglobulins, such as IgE, interferons suchas interferon α, interferon β or interferon γ, tumour necrosis factorTNF (formerly known as tumour necrosis factor-α), tumor necrosisfactor-β, colony stimulating factors such as G-CSF or GM-CSF, andplatelet derived growth factors such as PDGF-α, and PDGF-β and whereappropriate receptors thereof. Other antigens include bacterial cellsurface antigens, bacterial toxins, viruses such as influenza, EBV,HepA, B and C, bioterrorism agents, radionuclides and heavy metals, andsnake and spider venoms and toxins.

In one embodiment, the antibody may be used to functionally alter theactivity of the antigen of interest. For example, the antibody mayneutralize, antagonize or agonise the activity of said antigen, directlyor indirectly.

In a preferred embodiment the protein of interest expressed by the cellsaccording to the present invention is an anti-TNF antibody, morepreferably an anti-TNF Fab′, as described in WO01/094585 (the contentsof which are incorporated herein by reference).

Preferably the antibody molecule has specificity for human TNF (formerlyknown as TNFα), wherein the light chain comprises the light chainvariable region of SEQ ID NO: 11 and the heavy chain comprises the heavychain variable region of SEQ ID NO: 12.

Preferably the antibody molecule having specificity for human TNF is aFab′ and has a light chain sequence comprising or consisting of SEQ IDNO: 13 and a heavy chain sequence comprising or consisting of SEQ ID NO:14.

The inventors of the present invention have surprisingly discovered thatFab yield may be improved by expression in one or more cells accordingto the present invention. Without wishing to be bound by theory, themutated DegP gene used in the strains of the present invention havingchaperone activity and reduced protease activity improves Fab yieldbecause the chaperone activity of DegP facilitates the correct foldingof Fab.

After expression, antibody fragments may be further processed, forexample by conjugation to another entity such as an effector molecule.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins (such as enzymatically activetoxins of bacterial or plant origin and fragments thereof e.g. ricin andfragments thereof) biologically active proteins, for example enzymes,other antibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids and fragments thereof e.g. DNA, RNA andfragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy. Effector molecular may be attached to the antibody orfragment thereof by any suitable method, for example an antibodyfragment may be modified to attach at least one effector molecule asdescribed in WO05/003171 or WO05/003170 (the contents of which areincorporated herein by reference). WO05/003171 or WO05/003170 alsodescribe suitable effector molecules.

In one embodiment the antibody or fragment thereof, such as a Fab, isPEGylated to generate a product with the required properties, forexample similar to the whole antibodies, if required. For example, theantibody may be a PEGylated anti-TNF-α Fab′, as described inWO01/094585, preferably having attached to one of the cysteine residuesat the C-terminal end of the heavy chain a lysyl-maleimide-derived groupwherein each of the two amino groups of the lysyl residue has covalentlylinked to it a methoxypoly(ethyleneglycol) residue having a molecularweight of about 20,000 Da, such that the total average molecular weightof the methoxypoly(ethyleneglycol) residues is about 40,000 Da, morepreferably the lysyl-maleimide-derived group is[1-[[[2-[[3-(2,5-dioxo-1-pyrrolidinyl)-1-oxopropyl]amino]ethyl]amino]-carbonyl]-1,5-pentanediyl]bis(iminocarbonyl).

The cell may also comprise further polynucleotide sequences encoding oneor more further proteins of interest.

The polynucleotide encoding the protein of interest may be expressed asa fusion with another polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature polypeptide. The heterologous signal sequence selected should beone that is recognized and processed by the host cell. For prokaryotichost cells that do not recognize and process the native or a eukaryoticpolypeptide signal sequence, the signal sequence is substituted by aprokaryotic signal sequence. Suitable signal sequences include OmpA,PhoA, LamB, PelB, DsbA and DsbC.

In one embodiment an expression cassette is employed in the presentinvention to carry the polynucleotide encoding the protein of interestwhich typically comprises one or more protein coding sequences encodingone or more proteins of interest and one or more regulatory expressionsequences. The one or more regulatory expression sequences may include apromoter. The one or more regulatory expression sequences may alsoinclude a 3′ untranslated region such as a termination sequence.Suitable promoters are discussed in more detail below.

In one embodiment, the cell according to the present invention comprisesa vector, such as plasmid. The vector preferably comprises one or moreof the expression cassettes as defined above.

The vector for use in the present invention may be produced by insertingan expression cassette as defined above into a suitable vector.Alternatively, the regulatory expression sequences for directingexpression of the polynucleotide sequence encoding a protein of interestmay be contained in the vector and thus only the encoding region of thepolynucleotide may be required to complete the vector.

Examples of vectors which may be employed to transform the host cellwith a polynucleotide according to the invention include:

-   -   a plasmid, such as pBR322 or PACYC184, and/or    -   a viral vector such as bacterial phage;    -   a transposable genetic element such as a transposon.

Many forms of expression vector are available. Such vectors usuallycomprise a plasmid origin of DNA replication, an antibiotic selectablemarker a promoter and transcriptional terminator separated by amulti-cloning site (expression cassette) and a DNA sequence encoding aribosome binding site.

The promoters employed in the present invention can be linked to therelevant polynucleotide directly or alternatively be located in anappropriate position, for example in a vector such that when therelevant polypeptide is inserted the relevant promoter can act on thesame. In one embodiment the promoter is located before the encodingportion of the polynucleotide on which it acts, for example a relevantpromoter before each encoding portion of polynucleotide. “Before” asused herein is intended to imply that the promoter is located at the 5prime end in relation to the encoding polynucleotide portion.

The promoters may be endogenous or exogenous to the host cells. Suitablepromoters include Lac, tac, trp, PhoA, Ipp, Arab, Tet and T7.

One or more promoters employed may be inducible promoters.

Expression units for use in bacterial systems also generally contain aShine-Dalgarno (S. D.) ribosome sequence operably linked to the DNAencoding the polypeptide of interest.

In the embodiments of the present invention wherein a polynucleotidesequence comprises two or more encoding sequences for two or moreproteins of interest, for example an antibody light chain and antibodyheavy chain, the polynucleotide sequence may comprise one or moreinternal ribosome entry site (IRES) sequences which allows translationinitiation in the middle of an mRNA. An IRES sequence may be positionedbetween encoding polynucleotide sequences to enhance separatetranslation of the mRNA to produce the encoded polypeptide sequences.

The terminators may be endogenous or exogenous to the host cells. Asuitable terminator is rrnB.

Further suitable transcriptional regulators including promoters andterminators and protein targeting methods may be found in “Strategiesfor Achieving High-Level Expression of Genes in Escherichia coli” SavvasC. Makrides, Microbiological Reviews, September 1996, p. 512-538.

Embodiments of the invention described herein with reference to thepolynucleotide apply equally to alternative embodiments of theinvention, for example vectors, expression cassettes and/or host cellscomprising the components employed therein, as far as the relevantaspect can be applied to same.

According to a third aspect of the present invention there is provided amethod for producing a recombinant protein of interest comprisingexpressing the recombinant protein of interest in a recombinantgram-negative bacterial cell as described above in the first or secondaspect of the present invention.

The gram negative bacterial cell and protein of interest preferablyemployed in the method of the present invention are described in detailabove.

When the polynucleotide encoding the protein of interest is exogenousthe polynucleotide may be incorporated into the host cell using anysuitable means known in the art. Typically, the polynucleotide isincorporated as part of an expression vector which is transformed intothe cell. Accordingly, in one aspect the cell according to the presentinvention comprises an expression cassette comprising the polynucleotideencoding the protein of interest.

The polynucleotide sequence can be transformed into a cell usingstandard techniques, for example employing rubidium chloride, PEG orelectroporation.

The method according to the present invention may also employ aselection system to facilitate selection of stable cells which have beensuccessfully transformed with the polynucleotide encoding the protein ofinterest. The selection system typically employs co-transformation of apolynucleotide sequence encoding a selection marker. In one embodiment,each polynucleotide transformed into the cell further comprises apolynucleotide sequence encoding one or more selection markers.Accordingly, the transformation of the polynucleotide encoding theprotein of interest and the one or more polynucleotides encoding themarker occurs together and the selection system can be employed toselect those cells which produce the desired proteins.

Cells able to express the one or more markers are able tosurvive/grow/multiply under certain artificially imposed conditions, forexample the addition of a toxin or antibiotic, because of the propertiesendowed by the polypeptide/gene or polypeptide component of theselection system incorporated therein (e.g. antibiotic resistance).Those cells that cannot express the one or more markers are not able tosurvive/grow/multiply in the artificially imposed conditions. Theartificially imposed conditions can be chosen to be more or lessvigorous, as required.

Any suitable selection system may be employed in the present invention.Typically the selection system may be based on including in the vectorone or more genes that provides resistance to a known antibiotic, forexample a tetracycline, chloramphenicol, kanamycin or ampicillinresistance gene. Cells that grow in the presence of a relevantantibiotic can be selected as they express both the gene that givesresistance to the antibiotic and the desired protein.

In one embodiment, the method according to the present invention furthercomprises the step of culturing the transformed cell in a medium tothereby express the protein of interest.

An inducible expression system or a constitutive promoter may be used inthe present invention to express the protein of interest. Suitableinducible expression systems and constitutive promoters are well knownin the art.

Any suitable medium may be used to culture the transformed cell. Themedium may be adapted for a specific selection system, for example themedium may comprise an antibiotic, to allow only those cells which havebeen successfully transformed to grow in the medium.

The cells obtained from the medium may be subjected to further screeningand/or purification as required. The method may further comprise one ormore steps to extract and purify the protein of interest as required.

The polypeptide may be recovered from the strain, including from thecytoplasm, periplasm, or culture medium.

The specific method (s) used to purify a protein depends on the type ofprotein. Suitable methods include fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reversed-phase HPLC;hydrophobic-interaction chromatography; chromatography on silica;chromatography on an ion-exchange resin such as S-SEPHAROSE and DEAE;chromatofocusing; ammonium-sulfate precipitation; and gel filtration.

Antibodies may be suitably separated from the culture medium and/orcytoplasm extract and/or periplasm extract by conventional antibodypurification procedures such as, for example, protein A-Sepharose,protein G chromatography, protein L chromatography, thiophilic, mixedmode resins, Histag, FLAGTag, hydroxylapatite chromatography, gelelectrophoresis, dialysis, affinity chromatography, Ammonium Sulphate,ethanol or PEG fractionation/precipitation, ion exchange membranes,expanded bed adsorption chromatography (EBA) or simulated moving bedchromatography.

The method may also include a further step of measuring the quantity ofexpression of the protein of interest and selecting cells having highexpression levels of the protein of interest.

One or more method steps described herein may be performed incombination in a suitable container such as a bioreactor.

EXAMPLES Example 1 Generation of Mutant E. coli Cell Strains

The host cell strain used was W3110 genotype: F-LAM-IN (rrnD-rrnE)1 rph1 (ATCC no. 27325).

W3110A, as shown in the figures, is a different batch of W3110.

The following mutant E. coli cell strains were generated using a genereplacement vector system using the pKO3 homologousrecombination/replacement plasmid (Link et al., 1997, Journal ofBacteriology, 179, 6228-6237).

Mutant E. coli Cell Strain Genotype MXE001 ΔTsp MXE004 ΔTsp, ΔproteaseIII MXE005 ΔTsp, DegP S210A

Strain MXE001 was deposited on 21 May 2009 at the National Collection ofType Cultures, HPA, United Kingdom, under Accession number NCTC13444.

Strain MXE004 was deposited on 21 May 2009 at the National Collection ofType Cultures, HPA, United Kingdom, under Accession number NCTC13447.

Strain MXE005 was deposited on 21 May 2009 at the National Collection ofType Cultures, HPA, United Kingdom, under Accession number NCTC13448.

The Tsp, protease III and DegP integration cassettes were moved as SalI, Not I restriction fragments into similarly restricted pKO3 plasmids.

The plasmid uses the temperature sensitive mutant of the pSC101 originof replication (RepA) along with a chloramphenicol marker to force andselect for chromosomal integration events. The sacB gene which encodesfor levansucrase is lethal to E. coli grown on sucrose and hence (alongwith the chloramphenicol marker and pSC101 origin) is used to force andselect for de-integration and plasmid curing events. This methodologyhad been described previously (Hamilton et al., 1989, Journal ofBacteriology, 171, 4617-4622 and Blomfield et al., 1991, MolecularMicrobiology, 5, 1447-1457). The pKO3 system removes all selectivemarkers from the host genome except for the inserted gene.

The following plasmids were constructed.

pMXE191 comprising the knockout mutated Tsp gene as shown in the SEQ IDNO: 3 comprising EcoR I and Ase I restriction markers.

pMXE192 comprising the knockout mutated Protease III gene as shown inthe SEQ ID NO: 6 comprising EcoR I and Ase I restriction markers.

pMXE192 comprising the mutated DegP gene as shown in the SEQ ID NO: 9comprising an Ase I.

These plasmids were then transformed into chemically competent E. coliW3110 cells prepared using the method found in Chung C T et alTransformation and storage of bacterial cells in the same solution. PNAS86:2172-2175 (1989).

Day 1

40 μl of E. coli cells were mixed with (10 pg) 1 μl of pKO3 DNA in achilled BioRad 0.2 cm electroporation cuvette before electroporation at2500V, 25 μF and 200 Ω. 1000 μl of 2×PY was added immediately, the cellsrecovered by shaking at 250 rpm in an incubator at 30° C. for 1 hour.Cells were serially 1/10 diluted in 2×PY before 100 μl aliquots wereplated out onto 2×PY agar plates containing chloramphenicol at 20 μg/mlprewarmed at 30° C. and 43° C. Plates were incubated overnight at 30° C.and 43° C.

Day 2

The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation whilst colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2×PY. 100 μl of thiswas plated out onto 2×PY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C.

Day 3

Colonies here represent potential simultaneous de-integration andplasmid curing events. If the de-integration and curing events happenedearly on in the growth, then the bulk of the colony mass will be clonal.Single colonies were picked and replica plated onto 2×PY agar thatcontained either chloramphenicol at 20 μg/ml or 5% (w/v) sucrose. Plateswere incubated overnight at 30° C.

Day 4

Colonies that both grow on sucrose and die on chloramphenicol representpotential chromosomal replacement and plasmid curing events. These werepicked and screened by PCR with a mutation specific oligonucleotide.Colonies that generated a positive PCR band of the correct size werestruck out to produce single colonies on 2×PY agar containing 5% (w/v)sucrose and the plates were incubated overnight at 30° C.

Day 5

Single colonies of PCR positive, chloramphenicol sensitive and sucroseresistant E. coli were used to make glycerol stocks, chemicallycompetent cells and act as PCR templates for a PCR reaction with 5′ and3′ flanking oligos to generate PCR product for direct DNA sequencingusing Taq polymerase.

Cell strains MXE001, MXE004 and MXE005 were tested to confirm successfulmodification of genomic DNA carrying one or more the mutated proteasegenes by PCR amplification of the region of each mutated protease genecomprising a non-naturally occurring Ase I restriction site, as shown inFIGS. 1a, 1b and 1c , using oligonucleotides primers. The amplifiedregions of the DNA were then analyzed by gel electrophoresis before andafter incubation with Ase I restriction enzyme to confirm the presenceof the non-naturally occurring Ase I restriction site in the mutatedgenes. This method was carried out as follows:

The following oligos were used to amplify, using PCR, genomic DNA fromprepared E. coli cell lysates from MXE001, MXE004, MXE005, and W3110:

6284 Tsp 3′ (SEQ ID NO: 15) 5′-GCATCATAATTTTCTTTTTACCTC-3′ 6283 Tsp 5′(SEQ ID NO: 16) 5′-GGGAAATGAACCTGAGCAAAACGC-3′ 6362 Protease III 3'(SEQ ID NO: 17) 5′-GTGCCAGGAGATGCAGCAGCTTGC-3′ 6361 Protease III 5'(SEQ ID NO: 18) 5′-TTTGCAGCCAGTCAGAAAGTG-3′ 6282 DegP 5' (SEQ ID NO: 19)5′-CTGCCTGCGATTTTCGCCGGAACG-3′ 6281 DegP 3′ (SEQ ID NO: 20)5′-CGCATGGTACGTGCCACGATATCC-3′

The lysates were prepared by heating a single colony of cells for 10minutes at 95° C. in 20 μl of 1×PCR buffer. The mixture was allowed tocool to room temperature then centrifugation at 13,200 rpm for 10minutes. The supernatant was removed and labeled as ‘cell lysate’.

Each strain was amplified using every pair of oligos' Tsp pair, ProteaseIII pair and DegP pair.

The DNA was amplified using a standard PCR procedure.

 5 ul Buffer x10 (Roche)  1 ul dNTP mix (Roche, 10 mM mix) 1.5 ul 5′oligo (5 pmol) 1.5 ul 3′ oligo (5 pmol)  2 ul Cell lysate 0.5 ul Taq DNApolymerase (Roche 5 U/ul) 38.5 ul  H2OPCR Cycle.

94° C. 1 minute   94° C. 1 minute) 55° C. 1 minute) repeated for 30cycles 72° C. 1 minute) 72° C. 10 minutes 

Once the reactions were complete 25 ul was removed to a new microfugetube for digestion with Ase I. To the 25 ul of PCR reaction 19 ul ofH2O, 5 ul of buffer 3 (NEB), 1 ul of Ase I (NEB) was added, mixed andincubated at 37° C. for 2 hours.

To the remaining PCR reaction 5 ul of loading buffer (×6) was added and20 ul was loaded onto a 0.8% TAE 200 ml agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane.

Once the Ase I digestions were complete 10 ul of loading buffer (×6) wasadded and 20 ul was loaded onto a 0.8% TAE agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane.

Both gels were visualized using UV transluminator.

All genomic fragments amplified showed the correct sized band of 2.8 Kbfor Tsp, 1.8 Kb for protease III and 2.2K.b for DegP.

Following digestion with Ase I this confirmed the presence of theintroduced Ase I sites in the protease deficient strains but not in theW3110 control.

MXE001: genomic DNA amplified using the Tsp primer set and the resultingDNA was digested with Ase I to produce 2.2 and 0.6 Kbps bands.

MXE004: genomic DNA amplified using the Tsp primer set and the proteaseIII primer set and the resulting DNA was digested with Ase I to produce2.2 and 0.6 Kbps bands (Tsp fragments) and 1.0 and 0.8 Kbps bands(Protease III fragments).

MXE005 genomic DNA amplified using the Tsp primer set and the DegPprimer set and the resulting DNA was digested with Ase I to produce 2.2and 0.6 Kbps bands (Tsp fragments) and 1.25 and 0.95 Kbps bands (DegPfragments).

W3110 PCR amplified DNA was not digested by Ase I restriction enzyme.

Plasmid pMXE117 (pTTO CDP870 or 40.4), an expression vector for theCDP870 Fab′ (an anti-TNF Fab′), was constructed using conventionalrestriction cloning methodologies which can be found in Sambrook et at1989, Molecular cloning: a laboratory manual. CSHL press, N.Y. Theplasmid pMXE117 (pTTO CDP870 or 40.4) contained the following features;a strong tac promoter and lac operator sequence. The Fab light and heavychain genes were transcribed as a single dicistronic message. DNAencoding the signal peptide from the E. coli OmpA protein was fused tothe 5′ end of both light and heavy chain gene sequences, which directedthe translocation of the polypeptides to the E. coli periplasm.Transcription was terminated using a dual transcription terminator rrnBt1t2. The lacIq gene encoded the constitutively expressed Lac Irepressor protein. This repressed transcription from the tac promoteruntil de-repression was induced by the presence of allolactose or IPTG.The origin of replication used was p15A, which maintained a low copynumber. The plasmid contained a tetracycline resistance gene forantibiotic selection.

pMXE117 was then transformed into chemically competent proteasesdeficient cells (strains MXE001, MXE004 and MXE005) and W3110 cellsprepared using the method found in Chung C. T et at Transformation andstorage of bacterial cells in the same solution. PNAS 86:2172-2175(1989).

Example 2 Expression of an Anti-TNFα Fab′ in Mutated E. coli StrainsUsing Shake Flask Cultures

Strains MXE001, MXE004 and MXE005 were tested in a shake flaskexperiment comparing growth and expression of an anti-TNFα Fab′ againstW3110.

The shake flask experimental protocol used was performed as follows:

Preparation of Defined Medium Adapted Cells.

A single colony was picked into 5 ml 2×PY (1% phytone, Difco, 0.5% yeastextract, Difco, 0.5% NaCl) broth plus tetracycline (Sigma) at 10 ug/mland grown overnight at 37° C. with shaking at 250 rpm. 100 ul of thisovernight culture was used to inoculate 200 ml of chemically definedSM6E medium (described in Humphreys et al., 2002, Protein Expression andPurification, 26, 309-320) plus tetracycline at 10 ug/ml, grownovernight at 30° C. with shaking at 250 rpm. 100 ul of this secondovernight culture was used to inoculate a 2nd 200 ml SM6E media flaskplus tetracycline at 10 ug/ml. This was grown until the culture reachedan OD600 of about 2. The cultures were centrifuged briefly to collectcells before being resuspended in 100 ml of SM6E. Glycerol was added toa final concentration of 12.5% before storing aliquots of ‘adaptedcells’ at −80° C.

200 ml Shake Flask Experiment

Shake flask cultures were initiated by addition of a 2 ml aliquot ofthawed defined medium ‘adapted cells’ to 200 ml of SM6E media plustetracycline 10 ug/ml. These where grown overnight at 30° C. withagitation at 250 rpm. Each strain being tested was grown in triplicate.

Cultures grown to 2.0 OD600 were induced for production of heterologousprotein by the addition of IPTG to 200 uM. 1 ml culture samples weretaken at 1 hr, 2 hr, 4 hr, 6 hr, 12 hr and 24 hrs and aftercentrifugation at 13,200 rpm for 5 minutes the cell pellet wasresuspended in 200 ul of periplasmic extraction buffer (100 mMTris.Cl/10 mM EDTA pH 7.4). Periplasmic extracts were agitated at 250rpm over night at 30° C. The next day, the extracts were centrifuged for10 minutes at 13,200 rpm, the supernatant decanted off and stored at−20° C. as ‘periplasmic extract’. The spent cell pellet was discarded.

ELISA Quantification.

96 well ELISA plates were coated overnight at 4° C. with AB141 (rabbitanti-human CH1, UCB) at 2 μmml⁻¹ in PBS. After washing 3× with 300 ul ofsample/conjugate buffer (PBS, BSA 0.2% (w/v), Tween 20 0.1% (v/v)),serial ½ dilutions of samples and standards were performed on the platein 100 μl of sample/conjugate buffer, and the plate agitated at 250r.p.m at room temperature for 1 hour. After washing 3× with 300 ul ofwash buffer (PBS, Tween 20 0.1% (v/v)), 100 μl of the revealing antibody6062 (rabbit anti-human kappa HRP conjugated, The Binding Site,Birmingham, U.K.) was added, after dilution at 1/1000 insample/conjugate buffer. The plate was then agitated at 250 r.p.m atroom temperature for 1 hour. After washing with 3×300 ul of wash buffer,100 μl of TMB substrate was added (50:50 mix of TMB solution(Calbiochem): dH₂O) and the A₆₃₀ recorded using an automated platereader. The concentration of Fab′ in the periplasmic extracts werecalculated by comparison with purified Fab′ standards of the appropriateisotype.

FIG. 2 shows growth of MXE005 and MXE001 compared to the wild typeW3110.

FIG. 3 shows improved expression of the Fab′ from MXE005 and MXE001strains compared to the wild type W3110.

FIG. 4 shows the growth of MXE004 and W3110 and FIG. 5 shows expressionof the Fab′ in MXE004 and W3110 where it can be seen that the expressionfrom MXE004 was higher than W3110.

Example 3 Expression of an Anti-mIL13 Mouse Fab in Mutated E. coliStrains Using Shake Flask Cultures

Strains MXE001, MXE004, MXE005 and wild type W3110 cells weretransformed with plasmid pMKC006 expressing a murinised anti-mIL13 Fab′and tested using the same shake flask method described in Example 2except the experiment was stopped after 6 hours instead of 24 hours.

FIG. 6 shows the expression of an anti-mIL-13 mouse Fab in MXE001,MXE004, MXE005 and W3110, where it can be seen that MXE001, MXE004 andMXE005 show higher Fab expression compared to W3110.

Example 4 Analysis of Light and Heavy Chain Expression from Mutated E.coli Strains

Periplasmic extracts from strain MXE005 and wild type W3110 cellstransformed with plasmid pMXE117, from the shake flask experimentdescribed in Example 2 and were tested using a surface Plasmon resonancebinding assay performed using a BIAcore™ 2000 instrument (PharmaciaBiosensor AB, Uppsala, Sweden). The anti-TNFα Fab′ was immobilised ontoCM5 sensor chips using standard NHS/EDC chemistry. Residual NHS esterswere inactivated with ethanolamine hydrochloride (1 M).

Fab′ fragments were captured by either an immobilised monoclonalantiheavy chain or by an immobilised monoclonal anti-light chainantibody in separate flow cells. The presence of bound Fab′ was revealedby binding of the complementary monoclonal antibody (anti-light chain oranti-heavy chain) in a second step. High levels of immobilised antibodyensure that measurements are performed under mass transport-limitedconditions, where the contribution of the association rate constant tobinding is low in comparison to the contribution made by theconcentration of the Fab′ in the sample. The solution phase monoclonalantibody used in the second step is passed over the surface at a highconcentration so that binding is not limited by the association rateconstant of this interaction.

Assembled Fab′ fragments and correctly folded unassembled chains areboth detected during the first capture step. Binding of the secondantibody is only to an intact Fab′ fragment. Therefore, analysis of therelative binding at the first and second stages reveals the presence ofeither excess unassembled light chain, or excess unassembled heavy chainin the Fab′ sample and provides information on the stoichiometry ofassembly.

Assays were performed in both configurations for each sample, and eachsample was run in duplicate and in a randomised order.

-   -   (i) Where the concentration of assembled Fab′ was to be        determined by light chain capture, samples and standards zu at        10 gl/min) were injected over immobilised HP6053, followed by a        second step in which HP6045 at 300 Rg/ml was passed over the        surface in the solution phase.    -   (ii) Where the concentration of assembled Fab′ was to be        determined by heavy chain capture, samples and standards (10 t        at tOjuVmin) were injected over immobilised HP6045, followed by        a second step in which HP6053 at 5001 lg/ml was passed over the        surface in the solution phase. In both cases, the surface was        regenerated with 10 gi of 30 mM HCl at 30 l/min.

The number of resonance units determined using the BlAevaluation 3.1(Pharmacia Biosensor AB), was read against a standard curve.

FIG. 7 shows the light chain (L chain), heavy chain (H chain) and Fabexpression during the course of a fermentation run where a higher lightchain, heavy chain and Fab′ expression from MXE001 after 2 hours, 4hours and 6 hours compared to W3110 is shown. FIG. 7 shows higher lightchain after 6 hours from MXE005 compared to W3110 and higher Fab′expression from MXE005 after 2 hours, 4 hours and 6 hours compared toW3100.

Example 5 Analysis of Proteolysis Activity of Mutated E. coli Strainsfor Fab′

Periplasmic extracts from strains MXE001, MXE005 and wild type W3110cells, transformed with plasmid pMXE117 from the Shake flask experimentin Example 2 were tested in a polyclonal western blot analysis comparingproteolysis of an anti-TNFα Fab′ as follows:

12 ul of each periplasmic extract plus 4 ul of SDS-PAGE loading buffer(Invitrogen) was heated to 85° C. for 5 minutes, allowed to cool to 25°C. then centrifuged briefly before loading on to a pre prepared NuPAGE4-12% Bis-Tris gel (Invitrogen). SeeBlue 2 size markers (Invitrogen)were used for molecular weight estimation. The gel was electrophoresedfor 1 hour at 150V before transfer of proteins onto pre-wetted PVDFmembrane (Invitrogen) using immunoblotting at 150 mA for 2 hours. Themembrane was blocked for 1 hr in ‘blocking buffer’ (PBS, 3% (w/v) milkpowder, 0.1% (v/v) Tween20 (Sigma)) with gentle agitation. A polyclonalrabbit anti-human Fab′ sera (UCB) was applied at a dilution of 1 in 1000in 5 mls of blocking buffer and incubated at room temperature for 1 hourwith gentle agitation. The membrane was washed three times for 5 minseach with gentle agitation with blocking buffer. A secondary antibody(donkey anti-rabbit IgG HRP conjugated antibody (Jackson)) applied at adilution of 1 in 5000 in blocking buffer and incubation at roomtemperature for 1 hour with gentle agitation. The membrane was washedfour times for 5 minutes each with agitation firstly with blockingbuffer followed by PBS, 0.1% Tween for two washes then PBS for the finalwash. The blot was visualized using Metal Enhanced Dab substrate (ThermoScientific).

FIG. 8 shows the results of the western blot analysis where W=W3110,1=MXE001 (ΔTsp) and 5=MXE005 (ΔTsp, DegP S210A). Fragmentation aroundthe 14KDa is thought to represent proteolytic fragments of the lightchain of the expressed Fab′. It can be seen that MXE001 and MXE005 haveless proteolysed products compared to the wild type W3110 around the14KDa mark. Without being bound by theory, this data suggests that theanti-TNFα Fab′ is susceptible to proteolysis by Tsp and DegP.

Example 6 Growth of Mutated E. coli Strains and Expression of Fab′ inMutated E. coli Strains Using High Density Fermentations

Strain MXE005 and wild type W3110 cells were transformed with plasmidpMXE117 tested in fermentation experiments comparing growth andexpression of an anti-TNFα Fab′.

Growth Medium.

The fermentation growth medium was based on SM6E medium (described inHumphreys et al., 2002, Protein Expression and Purification, 26,309-320) with 3.86 g/l NaH₂PO₄.H₂O and 112 g/l glycerol.

Inoculum.

Inoculum cultures were grown in the same medium supplemented with 10μg/ml tetracycline. Cultures were incubated at 30° C. with agitation forapproximately 22 hours.

Fermentation.

Fermenters (2.5 liters total volume) were seeded with inoculum cultureto 0.3-0.5 OD₆₀₀. Temperature was maintained at 30° C. during the growthphase and was reduced to 25° C. prior to induction. The dissolved oxygenconcentration was maintained above 30% air saturation by variableagitation and airflow. Culture pH was controlled at 7.0 by automatictitration with 15% (v/v) NH₄OH and 10% (v/v) conc. H₂SO₄. Foaming wascontrolled by the addition of 10% (v/v) Struktol J673 solution (Schilland Seilacher).

A number of additions were made at different stages of the fermentation.When biomass concentration reached approximately 40 OD₆₀₀, magnesiumsalts and NaH₂PO₄.H₂O were added. Further additions of NaH₂PO₄.H₂O weremade prior to and during the induction phase to ensure phosphate wasmaintained in excess. When the glycerol present at the beginning offermentation had depleted (approximately 75 OD₆₀₀) a continuous feed of80% (w/w) glycerol was applied. At the same point in the fermentation anIPTG feed at 170 μM was applied. The start of IPTG feeding was taken asthe start of induction. Fermentations were typically run for 70-73 hoursat the lower glycerol feed rates (0.5-2.5 ml/h) and 50-60 h at thehigher glycerol feed rates (5.4-10.9 ml/h).

Measurement of Biomass Concentration and Growth Rate.

Biomass concentration was determined by measuring the optical density ofcultures at 600 nm.

Periplasmic Extraction.

Cells were collected from culture samples by centrifugation. Thesupernatant fraction was retained (at −20° C.) for further analysis. Thecell pellet fraction was resuspended to the original culture volume inextraction buffer (100 mM Tris-HCl, 10 mM EDTA; pH 7.4). Followingincubation at 60° C. for approximately 16 hours the extract wasclarified by centrifugation and the supernatant fraction retained (at−20° C.) for analysis.

Fab′ Quantification.

Fab′ concentrations in periplasmic extracts and culture supernatantswere determined by Fab′ assembly ELISA as described in Humphreys et al.,2002, Protein Expression and Purification, 26, 309-320.

FIG. 9 shows the growth profile of MXE001 compared to control W3110which shows that the growth profiles are substantially the same forapproximately 35 hours.

FIG. 10 shows Fab yield from the supernatant (dotted lines) andperiplasm (solid lines) from E. coli strain MXE001 compared to controlW3110. The MXE001 strain shows higher periplasmic Fab′ expression up toapproximately 30 hours and significantly higher supernatant Fab′expression over the whole fermentation period.

FIG. 11 shows the total Fab′ yield from the supernatant and periplasm ofthe E. coli strain MXE001 compared to control W3110 where it can be seenthat the MXE005 strain produced higher Fab′ yield compared to thecontrol W3110.

FIG. 12 shows the Fab′ specific production rate of E. coli strain MXE001compared to the control W3110 where it can be seen that MXE001 hassignificantly higher specific production rate compared to W3110.

FIG. 13 shows the growth profile of MXE004 and MXE005 compared tocontrol W3110. The growth profiles of MXE004 and MXE005 are faster overthe initial period of approximately 35 hours compared to the controlW3110.

FIG. 14 shows Fab′ yields from the supernatant (dotted lines) andperiplasm (solid lines) of E. coli strains MXE004, MXE005 and the W3110control. The MXE005 strain shows higher Fab′ yield from the periplasmfor approximately 28 hours compared to the control and significantlyhigher supernatant Fab′ yield compared to the control over the wholefermentation period. The MXE004 strain shows higher Fab′ yield from theperiplasm for approximately 20 hours compared to the control andsignificantly higher supernatant Fab′ yield compared to the control overthe whole fermentation period.

FIG. 15 shows the total Fab′ yield from the supernatant and periplasm ofthe E. coli strains MXE004 and MXE005 where it can be clearly seen thatthe MXE004 and MXE005 strains produced significantly higher yieldcompared to the control.

FIG. 16 shows the Fab′ specific production rate of E. coli strainsMXE004 and MXE005 and the W3110 control where it can be seen that MXE004and MXE005 have a significantly higher specific production rate comparedto W3110.

Example 7 Growth of Mutated E. coli Strains MXE001, MXE004 and MXE005Compared to W3110 and Highly Mutated E. coli Strains in Shake FlaskExperiment

The following strains were analyzed in a shake flask experiment toassess growth rate: Mutated E. coli strains MXE001, MXE004 and MXE005derived from W3110 (Example 1);

Wild-type E. coli strain W3110;

SURE (Stratagene) having genotype: endA1 glnV44 thi-1 gyrA96 relA1 lacrecB recJ sbcC umuC::Tn5 uvrC e14-Δ(mcrCB-hsdSMR-mrr)171 F′[proAB⁺lacI^(q) lacZΔM15 Tn10];

STBL3 (Invitrogen) having genotype: F-glnV44 recA13 mcrB mrr hsdS20(rB-,mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1;

TOP10 (Invitrogen) having genotype: F-mcrA Δ(mrr-hsdRMS-mcrBC);φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16rpsL(Str^(R)) endA1λ⁻; and

XL1-Blue (Stratagene) having genotype endA1 gyrA96(nal^(R)) thi-1 recA1relA1 lac glnV44 F′[::Tn10 proAB⁺ lacI^(q) Δ(lacZ)M15] hsdR17(r_(K)⁻m_(K) ⁺).

A single colony was picked into 5 ml of LB broth (10 g Tryptone, 5 gyeast extract, 10 g Na Cl per liter) and grown overnight at 37° C. withshaking at 250 rpm. The overnight culture was used to inoculate 75 ml ofLB broth to an OD₆₆₀ of 0.1 (n=2). The cultures were grown at 37° C.with shaking at 250 rpm, 0.2 ml samples were removed every hour and theOD₆₀₀ recorded. The OD600 was then plotted against time in hours and theresults are shown in FIG. 17. It can be seen from FIG. 17 that theheavily mutated E. coli strains have a lower growth rate compared toMXE001, MXE004, MXE005 and W3110.

While this invention has been particularly shown and described withreference to preferred embodiments, it will be understood to thoseskilled in the art that various changes in form and detail may be madewithout departing from the scope of the invention as defined by theappendant claims.

We claim:
 1. A method for producing a recombinant protein of interestcomprising expressing a polynucleotide encoding the recombinant proteinof interest in a recombinant gram-negative bacterial cell selected fromthe group consisting of: a) a mutant E. coli cell strain identified asMXE002 having genotype Δptr and deposited with the National Collectionof Type Cultures, HPA, United Kingdom, under Accession Number NCTC13445;b) a mutant E. coli cell strain identified as MXE003 having genotypeDegP S210A and deposited with the National Collection of Type Cultures,HPA, United Kingdom, under Accession Number NCTC13446; and c) a mutantE. coli cell strain identified as MXE006 having genotype Δptr, DegPS210A and deposited with the National Collection of Type Cultures, HPA,United Kingdom, under Accession Number NCTC13449.
 2. The methodaccording to claim 1, wherein said recombinant protein of interest is anantibody or antigen-binding fragment thereof.
 3. The method according toclaim 2, wherein said antibody or antigen-binding fragment thereofspecifically binds tumour necrosis factor (TNF).
 4. A method forproducing a recombinant protein of interest comprising expressing apolynucleotide encoding the recombinant protein of interest in arecombinant gram-negative bacterial cell comprising said polynucleotideencoding a protein of interest, a mutated DegP gene, wherein the mutatedDegP gene encodes a DegP protease that comprises a mutation at an aminoacid selected from the group consisting of His105, Asp135, Ser210 andcombinations thereof and said bacterial cell is isogenic to a wild-typebacterial cell except for the mutated DegP gene and, optionally, one ormore of the following genetic alterations: i) a mutated ptr gene,wherein the mutated ptr gene encodes a Protease III protein havingreduced protease activity or is a knockout mutated ptr gene; and/or ii)a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp proteinhaving reduced protease activity or is a knockout mutated Tsp gene. 5.The method according to claim 4, wherein the DegP protease comprises: amutation to His105; a mutation to Asp135; a mutation to Ser210; amutation to His105 and Asp135; a mutation to His105 and Ser210; amutation to Asp135 and Ser210; or a mutation to His105, Asp135 andSer210.
 6. The method according to claim 5, wherein one or more ofHis105, Asp135, and Ser210 are substituted with a glycine or alanine. 7.The method according to claim 5, wherein His105, Asp135, and Ser210 aresubstituted with a glycine or alanine.
 8. The method according to claim5, wherein His105 is substituted with a glycine, alanine, valine orleucine.
 9. The method according to claim 5, wherein Asp135 issubstituted with a glycine, alanine, lysine or arginine.
 10. The methodaccording to claim 5, wherein Ser210 is substituted with a glycine,alanine, valine, leucine, phenylalanine or tyrosine.
 11. The methodaccording to claim 5, wherein His105 is substituted with a glycine,alanine, valine or leucine; Asp135 is substituted with a glycine,alanine, lysine or arginine; and Ser210 is substituted with a glycine,alanine, valine, leucine, phenylalanine or tyrosine.
 12. The methodaccording to claim 4, wherein said bacterial cell comprises saidpolynucleotide encoding said protein of interest, a mutated DegP geneand a mutated ptr gene, wherein the mutated ptr gene encodes a ProteaseIII protein having reduced protease activity or is a knockout mutatedptr gene.
 13. The method according to claim 4, wherein said bacterialcell comprises a mutated DegP gene and a mutated Tsp gene, wherein themutated Tsp gene encodes a Tsp protein having reduced protease activityor is a knockout mutated Tsp gene.
 14. The method according to claim 4,wherein said bacterial cell comprises said polynucleotide encoding saidprotein of interest and a mutated DegP gene.
 15. The method according toclaim 4, wherein said bacterial cell comprises said polynucleotideencoding said protein of interest, a mutated DegP gene, a mutated ptrgene, wherein the mutated ptr gene encodes a Protease III protein havingreduced protease activity or is a knockout mutated ptr gene, and amutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp proteinhaving reduced protease activity or is a knockout mutated Tsp gene. 16.The method according to claim 4, wherein said protein of interest is anantibody or antigen-binding fragment thereof.
 17. The method accordingto claim 12, wherein said protein of interest is an antibody orantigen-binding fragment thereof.
 18. The method according to claim 13,wherein said protein of interest is an antibody or antigen-bindingfragment thereof.
 19. The method according to claim 14, wherein saidprotein of interest is an antibody or antigen-binding fragment thereof.20. The method according to claim 15, wherein said recombinant proteinof interest is an antibody or antigen-binding fragment thereof.
 21. Themethod according to claim 16, wherein said antibody or antigen-bindingfragment thereof specifically binds tumour necrosis factor (TNF). 22.The method according to claim 17, wherein said antibody orantigen-binding fragment thereof specifically binds tumour necrosisfactor (TNF).
 23. The method according to claim 18, wherein saidantibody or antigen-binding fragment thereof specifically binds tumournecrosis factor (TNF).
 24. The method according to claim 19, whereinsaid antibody or antigen-binding fragment thereof specifically bindstumour necrosis factor (TNF).
 25. The method according to claim 20,wherein said antibody or antigen-binding fragment thereof specificallybinds tumour necrosis factor (TNF).